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Distinct characteristics of amyloid deposits in early- and late-onset transthyretin Val30Met familial amyloid polyneuropathy
Journal of the Neurological Sciences 287 (2009) 178–184
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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
Distinct characteristics of amyloid deposits in early- and late-onset transthyretin Val30Met familial amyloid polyneuropathy Haruki Koike a, Yukio Ando b, Mitsuharu Ueda b, Yuichi Kawagashira a, Masahiro Iijima a, Junko Fujitake c, Michiyuki Hayashi c, Masahiko Yamamoto d, Eiichiro Mukai e, Tomohiko Nakamura a, Masahisa Katsuno a, Naoki Hattori a, Gen Sobue a,⁎ a
Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Diagnostic Medicine, Kumamoto University School of Medicine, Kumamoto, Japan c Department of Neurology, Kyoto City Hospital, Kyoto, Japan d Department of Speech Pathology and Audiology, Aichi Gakuin University School of Health Science, Aichi, Japan e Department of Neurology, Nagoya Medical Center, Nagoya, Japan b
a r t i c l e
i n f o
Article history: Received 25 March 2009 Rceived in revised form 29 July 2009 Accepted 31 July 2009 Available online 25 August 2009 Keywords: Familial amyloid polyneuropathy Amyloid Congo red staining Transthyretin Autopsy Surface enhanced desorption/ionization timeof-flight mass spectrometry
a b s t r a c t Late-onset transthyretin Val30Met-associated familial amyloid polyneuropathy (FAP ATTR Val30Met) cases unrelated to endemic foci in Japan show different clinicopathological features from the conventional earlyonset cases in endemic foci. We compared the characteristics of amyloid deposits in early-onset FAP ATTR Val30Met cases in endemic foci and late-onset cases in non-endemic areas. Amyloid deposits in three earlyonset cases from endemic foci and five late-onset cases from non-endemic areas were systematically examined post-mortem. Amyloid deposits in early-onset cases were highly congophilic and showed strong apple-green birefringence with Congo red staining and had long, parallel fibrils in most organs. On the other hand, those in late-onset cases were generally weakly congophilic and showed faint apple-green birefringence with Congo red staining and had short, haphazard fibrils. In the renal glomus and adrenal gland of early-onset cases, the characteristics of amyloid deposits were similar to those observed in lateonset cases. Analysis of cardiac amyloid using surface enhanced desorption/ionization time-of-flight mass spectrometry indicated that most transthyretin (TTR) was variant in early-onset cases, while more than half was composed of wild-type TTR in late-onset cases. Although characteristics of amyloid deposits may differ among individual organs of respective cases, especially in early-onset cases, the pattern was distinct between early- and late-onset cases. Amyloid deposition in late-onset cases may be similar to that observed in senile systemic amyloidosis with wild-type TTR deposition, suggesting that aging may play an important role in these cases. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Transthyretin Val30Met-associated familial amyloid polyneuropathy (FAP ATTR Val30Met) is the most common type of familial amyloid polyneuropathy [1–9]. The classical concept of this disease was that most patients have a relationship to endemic foci in Japan, Portugal, and Sweden [2–4,6–8]. However, through the development of gene diagnostic techniques, many patients unrelated to these endemic foci have been reported in many nations worldwide [5,9–12], suggesting that this disease is more prevalent than was generally believed. Previous studies of Japanese FAP ATTR Val30Met patients
⁎ Corresponding author. Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. Tel.: +81 52 744 2385; fax: +81 52 744 2384. E-mail address: [email protected] (G. Sobue). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.07.028
have demonstrated significant differences between patients in endemic foci and those in non-endemic areas [10,11,13,14]. Although there are exceptions, typical cases of FAP ATTR Val30Met in these endemic foci show characteristic features including an age at onset from the late 20 s to the early 40 s, a high penetrance rate, marked autonomic dysfunction, loss of superficial sensation including nociception and thermal sensation (i.e. sensory dissociation), atrioventricular conduction block requiring pacemaker implantation, and anticipation of the age at onset [2,3,8,11,15,16]. On the other hand, patients in non-endemic areas usually show an older age at onset over 50 years, a low penetrance rate, relatively mild autonomic dysfunction, loss of all sensory modalities rather than sensory dissociation, frequent presence of cardiomegaly, extreme male preponderance, and an absence of anticipation of the age at onset [10,11,13,14,16,17]. Differences in the geographical distribution and clinical features between the early- and late-onset types of FAP ATTR Val30Met have also been reported in Portugal [6,18,19]. The causal mechanism of the
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Table 1 Background of patients with early- and late-onset FAP ATTR Val30Met. Age at onset/ death (years)
Initial symptom
Sensory dissociation
Cardiac involvement Cardio-megalya
Pacemaker implantation
Early-onset casesb 1 F 2 F 3 M
28/35 37/51 24/41
A A A
+ + +
− − −
+ + −
Sudden death Pneumonia Pneumonia
Late-onset casesc 4 5 6 7 8
64/67 62/68 52/62 59/74 61/66
P P P P P
+ + − + −
− + + − +
− − − − −
Lung cancer Heart failure Heart failure Heart failure Heart failure
Patients
Sex
M M M M M
Cause of death
+ = present; − = absent; F = female; M = male; A = autonomic symptoms; P = paresthesia in the legs. a The presence of cardiomegaly was assessed at the time of first referral to the hospital. b Early-onset FAP ATTR Val30Met cases in endemic foci. c Late-onset FAP ATTR Val30Met cases in non-endemic areas.
differences between the early- and late-onset types of FAP with the same mutation in the TTR gene has not yet been determined. In this context, we systematically investigated and compared the characteristics of amyloid deposits in tissues from autopsied patients with conventional early-onset FAP ATTR Val30Met from endemic foci and late-onset FAP ATTR Val30Met unrelated to these endemic foci.
sections. All specimens from all cases were stained simultaneously for each method. For electron microscopy, formalin-fixed, paraffinembedded tissue sections were deparaffinized and embedded in epoxy resin. Epoxy-resin embedded specimens were cut into ultrathin sections and stained with uranyl acetate and lead citrate.
2. Materials and methods 2.1. Patients Eight autopsied cases with genetically-confirmed FAP ATTR Val30Met were examined: three early-onset patients (1 male and 2 females) from endemic foci; and five late-onset patients (5 males) from non-endemic areas of Japan. No patient in the study belonged to the same kindred as another. Informed consent was obtained from the patient's families, and all aspects of the study were approved by the Ethics Committees of Nagoya University Graduate School of Medicine. The characteristics of the patients are summarized in Table 1. Although the duration of the disease was longer in the early-onset cases (12.7 ± 5.1 years for early-onset cases and 7.8 ± 4.8 years for late-onset cases, mean ± SD), heart weight was heavier in the lateonset cases (437 ± 15 g in early-onset cases and 611 ± 160 g in lateonset cases). In the late-onset cases, the enlargement of the whole heart was evident and ventricular walls were markedly hypertrophic, except for case 4 whose duration of the disease was shortened due to lung cancer. Therefore, the cause of death in the late-onset cases was mainly due to heart failure. The pathological findings, focusing on the distribution of amyloid deposits in various organs in 7 of these cases (patients 1 to 7), have been reported previously [13,20]. 2.2. Pathological examinations The nervous system including the brain, spinal cord, ventral and dorsal roots, dorsal root ganglia from L3 to L5, sympathetic ganglia, proximal portion of the sciatic nerve, and visceral organs were removed, fixed in 10% formalin solution, and embedded in paraffin [13,21,22]. All specimens were cut into 4 μm thick sections by routine methods and stained with hematoxylin and eosin and alkaline Congo red. Congo red-stained sections were viewed under polarized light. The degree of the intensity for glittering birefringence after Congo red staining was quantified into three levels as follows: 1, low; 2, middle ; and 3, high. Immunohistochemical assessment was performed with a peroxidase-antiperoxidase method using an anti-human transthyretin (TTR) antibody (Dako; 1:1000) in consecutive deparaffinized
Table 2 Intensity of birefringence after Congo red staining and morphology of amyloid fibrils. Early-onset casesa (n = 3)
Late-onset casesb (n = 5) Congo redc
Morphologyd
Long, parallel
1.0
ND
ND
1.0
Thyroid gland
3.0
Long, parallel
1.6
Lung
3.0
Long, parallel
1.0
Gastrointestinal tract Spleen
3.0
Long, parallel
1.0
2.7
Long, parallel
1.0
Pancreas
3.0
Long, parallel
1.2
Adrenal gland
1.3
Short, haphazard
ND
Short, haphazard Short, haphazard Short, haphazard Short, haphazard Short, haphazard Short, haphazard Short, haphazard ND
Kidney Cortex
1.0
ND
ND
3.0
Short, haphazard Long, parallel
1.5
Short, haphazard
2.7
Long, parallel
1.3
Dorsal root ganglia 2.3
Long, parallel
1.0
Sympathetic ganglia
Long, parallel
1.0
Short, haphazard Short, haphazard Short, haphazard
Congo redc
Morphology
Heart
3.0
Hypophysis
Medulla Nervous system Sciatic nerve
2.3
d
ND = not determined due to little amyloid deposition. a Early-onset FAP ATTR Val30Met cases in endemic foci. b Late-onset FAP ATTR Val30Met cases in non-endemic areas. c The degree of the intensity for glittering birefringence after Congo red staining was quantified into three levels as follows: 1, low; 2, middle ; and 3, high. Values were expressed as the mean. d Morphology of amyloid fibrils was determined using electron microscopy.
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Fig. 1. Representative characteristics of amyloid deposits in the heart from early-onset (A to D) and late-onset cases (E to H). (A, B, E, and F) Alkaline Congo red staining. (C and G) Immunostaining with the anti-human transthyretin (TTR) antibody using consecutive sections for Congo red staining. (D and H) Morphology of amyloid fibrils as observed with electron microscopy. By Congo red staining, amyloid deposits in early-onset cases were highly congophilic (A) and had bright and glittering apple-green birefringence when examined by polarized light (B), while those in late-onset cases were weakly congophilic (E) and showed faint birefringence under polarized light (F). Affinity to the anti-TTR antibody was strong in early-onset cases (C), while it was weak in late-onset cases (G). By electron microscopy, straight long fibrils that were often aligned in a parallel fashion were observed in early-onset cases (D), while fairly short and haphazardly arranged fibrils were present in late-onset cases (H). Bar = 0.3 μm.
2.3. Analysis of TTR in amyloid deposits To determine the proportion of variant (Val30Met) TTR in total (Val30Met plus wild-type) TTR in amyloid deposits, we used surface enhanced desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Amyloid deposits in the hearts from three earlyonset cases (patients 1 to 3) and four late-onset cases (patients 4 to 7) were analyzed. Tissue-deposited TTR was extracted in 6 M guanidine hydrochloride in 0.15 M Tris-HCl buffer, pH 8.0, from formalin-fixed paraffin sections as described previously [23]. Samples were purified by means of reversed-phase HPLC (Waters 600E HPLC system, Waters Co.) on an Aquapore C8 column (7 mm, 300 Å, 30× 4.6 mm, PerkinElmer), as described previously [23]. The collected TTR-rich fractions were dialyzed in 50 mM phosphate buffer, pH 6.0, and were used for SELDITOF-MS analyses. For SELDI-TOF MS, a strong anion exchange array with quaternary amine functionality (Q10) was used. Binding of proteins to ProteinChip surfaces was performed in a 96-well format bioprocessor (Bio-Rad Laboratories). Samples were analyzed with the PCS 4000 SELDI-TOF-MS instrument (Bio-Rad Laboratories). ProteinChips were read by using the following protocol: ion focus mass, 13,800 m/z; laser energy, 2000 nJ; matrix attenuation, 500 m/z; sample rate, 800; shots/pixel, 5; partition, 1 of 4; and acquired mass range from 0 to 100,000 m/z. External calibration of the instrument was performed by using the All-in-1 protein molecular mass standard (Bio-Rad Laboratories). Using the regression equation obtained in the quanti-
tative analysis of the ratio of variant to wild-type TTR via Q10 ProteinChip, we calculated the percentage of variant TTR in total TTR in amyloid deposits. To determine the peaks of variant TTR and wildtype TTR, sera from a normal subject and a heterozygous FAP ATTR Val30Met patient were also analyzed. 3. Results 3.1. Characteristics of amyloid deposits The characteristics of amyloid deposits in various organs are summarized in Table 2. As previously described [13], amyloid deposition was prominent in the heart, thyroid gland, gastrointestinal tract, pancreas, adrenal gland, kidney, and peripheral nervous system in early-onset cases. In late-onset cases, although the amount of amyloid deposits was generally less than that observed in early-onset cases, it was more conspicuous in the heart and the anterior lobe of the pituitary gland as compared to early-onset cases [13,20]. Because amyloid deposition was scarce, limited around small vessels, or absent in the parenchyma of the central nervous system, the posterior lobe of the pituitary gland, liver, and skeletal muscle in both groups, we did not assess the characteristics of amyloid deposits in these organs. When the sections were stained with alkaline Congo red, amyloid deposits in early-onset cases were highly congophilic and had bright and glittering apple-green birefringence when examined by polarized light (Table 2; Fig. 1A and B), except for those in the renal glomus and
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Fig. 2. Representative characteristics of amyloid deposits in the kidney from early-onset cases. (A and B) Alkaline Congo red staining. (C) Immunostaining with the anti-human transthyretin (TTR) antibody using consecutive sections for Congo red staining. (D and E) Morphology of amyloid fibrils as observed with electron microscopy. In A to C, the renal glomus is observed on the upper side of the photograph, while the medulla is located on the lower side. In the medulla, the affinity of amyloid deposits to Congo red was high (A, lower side) and showed bright apple-green birefringence (B, lower side), while it was weak (A, upper side) and showed faint birefringence (B, upper side) in the glomus. Affinity to the anti-TTR antibody was strong in the medulla (C, lower side), while it was weak in the glomus (C, upper side). By electron microscopy, straight long fibrils that were often aligned in a parallel fashion were observed in the medulla (D), while fairly short and haphazardly arranged fibrils were observed in the glomus (E). Bar = 0.3 μm.
adrenal gland. In the kidneys of early-onset cases, the affinity to Congo red of amyloid deposits was high and showed bright apple-green birefringence in the medulla, while affinity to Congo red of amyloid in the glomus was weak and showed faint birefringence (Fig. 2A and B). Interestingly, in the renal cortex, the affinity of amyloid to Congo red in the interstitium or vessel wall was high, showing a similar pattern to the other organs, despite its close proximity to the glomerular amyloid. Amyloid deposits in the adrenal gland also showed similar characteristics to those in the renal glomus. On the other hand, the amyloid deposits in the organs from late-onset cases were generally weakly congophilic and showed faint birefringence under polarized light as compared to those in most organs from the early-onset cases (Table 2; Fig. 1E and F). The intensity of TTR immunostaining of amyloid deposits showed similar tendency to the affinity to Congo red; it was largely higher in the early-onset cases than in the lateonset cases in most organs (Fig. 1C and G). Amyloid deposits in the renal glomus and adrenal gland of early-onset cases tended to show weak affinity to the anti-TTR antibody, which is similar to that observed in the late-onset cases (Fig. 2C). Electron microscopy revealed two patterns of amyloid fibrils corresponding well to the affinity to Congo red (Table 2). In amyloid deposits with a high affinity to Congo red seen in most organs in earlyonset cases, straight long fibrils that were often aligned in a parallel fashion were observed (Figs. 1D and 2D). On the other hand, fairly short and haphazardly arranged fibrils were present in the amyloid deposits with a low affinity to Congo red that were seen in the organs from late-onset cases and in the renal glomus and adrenal gland in early-onset cases (Figs. 1H and 2E). 3.2. The ratio of variant to wild-type TTR in amyloid deposits Fig. 3 shows SELDI-TOF mass spectra of TTR prepared from cardiac amyloid in early- and late-onset cases. A peak in early-onset cases showed a molecular mass of 13,792 m/z, corresponding to the variant TTR. On the other hand, two peaks were observed in the late-onset cases; one of which showed a molecular mass of 13,792 m/z,
corresponding to the variant TTR, and another showed 13,761 m/z, corresponding to the wild-type TTR, which was not detected in the early-onset cases. The percentage of variant TTR in total TTR in amyloid deposits was seen to be 42.3%, 41.4%, 37.1%, and 44.0% in patients 4 to 7, respectively. Because the peak of wild-type TTR was not detected in any early-onset cases, we considered the percentage of variant TTR in these cases to be more than 80%. 4. Discussion In this study, we examined the characteristics of amyloid deposits in patients with FAP ATTR Val30Met in Japan. By comparing the characteristics of Congo red staining and morphology of amyloid fibrils in conventional early-onset cases from endemic foci to those in late-onset cases from non-endemic areas, we demonstrated clear differences of amyloid deposits between these two types of FAP ATTR Val30Met. Amyloid deposits in early-onset cases tended to be highly congophilic and showed strong apple-green birefringence in Congo red staining and had long, parallel fibrils in most organs. On the other hand, those in late-onset cases were generally weakly congophilic and showed faint apple-green birefringence in Congo red staining and had short, haphazard fibrils. The intensity of TTR immunostaining also was different between early- and late-onset cases; it was largely higher in the early-onset cases than in the late-onset cases in most organs. We have to keep in mind the reproducibility of staining properties of these methods with caution. To minimize the ambiguity, we stained all sections simultaneously for each method. Also, we admit formalinfixed samples to be less suitable than glutaraldehyde-fixed samples for electron microscopic examination. However, by using formalinfixed samples, we were able to examine many sections of various organs. It enabled us to discover an entire spectrum of the morphology of amyloid fibrils. To confirm the difference between early- and late-onset cases, we used SELDI-TOF MS, which showed distinct characteristics of amyloid deposits. Deposited TTR in the heart was mostly variant in early-onset cases, while abundant wild-type TTR contributed in late-onset cases.
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Fig. 3. Mass spectra of transthyretin (TTR) prepared from cardiac amyloid in early-onset cases (patients 1 to 3) and late-onset cases (patients 4 to 7) by surface enhanced desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF MS). To determine the peaks of variant (Val30Met) TTR and wild-type TTR, sera from a normal subject and a heterozygous FAP ATTR Val30Met patient were also analyzed. A peak in early-onset cases showed a molecular mass of 13,792 m/z (arrowheads), corresponding to the variant TTR. On the other hand, two peaks were observed in late-onset cases; one of which showed a molecular mass of 13,792 m/z (arrowheads), corresponding to the variant TTR, and another showed 13,761 m/z (asterisks), corresponding to the wild-type TTR, which was not detected in early-onset cases. The percentage of variant TTR in total TTR in amyloid deposition was seen to be 42.3%, 41.4%, 37.1%, and 44.0% in patients 4 to 7, respectively. Because the peak of wild-type TTR was not detected in any of the early-onset cases, we considered the percentage of variant TTR in these cases to be more than 80%.
This abundance of wild-type TTR in late-onset cases may support the view that the mechanism of amyloid deposition is similar, to some extent, to that observed in senile systemic amyloidosis (SSA). In SSA, wild-type TTR is known to be an amyloid precursor protein [24]. SSA predominantly affects men [25,26]. The presence of an extreme male preponderance in late-onset FAP ATTR Val30Met in non-endemic areas, as opposed to a nearly 1 to 1 male–female ratio in early-onset cases in endemic foci, is also similar to that observed in SSA [10,11,26]. In addition, amyloid deposition in the heart and anterior pituitary gland, that characterized the late-onset FAP ATTR Val30Met cases in non-endemic areas, is sometimes found in the normal elderly population [27–30]. These findings suggest the importance of unidentified factors that change with aging to the amyloid deposition in late-onset FAP ATTR Val30Met cases. A previous study of cardiac amyloid deposits in Swedish patients with FAP ATTR Val30Met and SSA have demonstrated two distinct
patterns of amyloid deposition; one pattern is similar to the amyloid deposits from our early-onset cases, while the other pattern resembles those from our late-onset cases [31]. Swedish FAP ATTR Val30Met patients showed the former pattern in 6 of 11 patients and the latter pattern in 5 of 11 patients, while all SSA cases showed the latter pattern [31]. A recent study analyzing amyloid deposits in subcutaneous adipose tissue from Swedish FAP ATTR Val30Met patients revealed that the former pattern tended to be present in patients with an early-age at onset, while the latter pattern was detected in relatively late-age at onset patients [32]. The features of FAP ATTR Val30Met in a Swedish endemic focus are not similar to those in Japanese and Portuguese endemic foci but rather resemble those in late-onset cases from non-endemic areas of Japan i.e. the mean age at onset in the fifth decade of life, a low penetrance rate, a tendency to manifest sensori-motor neuropathy rather than autonomic symptoms as an initial symptom, and extensive amyloid
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deposition in the heart [1,4]. In some Swedish FAP ATTR Val30Met patients, symptoms of cardiac failure are more prominent than those of neuropathy [32]. The age at onset in these patients tend to be elderly and amyloid deposits in such patients show characteristics similar to those in SSA [31,32]. In addition, a recent epidemiological study showed the presence of a new endemic focus (Ishikawa prefecture) outside the conventional endemic foci (Arao and Ogawa) in Japan [33]. Features of patients in this new focus are different from those in the conventional endemic foci, but resemble those in Sweden [33]. Therefore, FAP ATTR Val30Met patients in Japan could now be classified into: (1) conventional early-onset cases in endemic foci (Arao and Ogawa); (2) late-onset cases in an endemic focus (Ishikawa); and (3) late-onset cases in non-endemic areas. In this context, cases in a Swedish endemic focus and Ishikawa prefecture seem to be an intermediate form between cases in nonendemic areas and those in other endemic foci. Because the late-onset type of FAP ATTR Val30Met in non-endemic areas is considered to be more prevalent in Japan and elsewhere than was generally appreciated [11,14], it may be hypothesized that it is a prototype of FAP ATTR Val30Met. Some indigenous environmental or genetic factors may have been created endemic foci and some of them induced anticipation creating the early-onset type of FAP ATTR Val30Met. Another interesting finding is that the characteristic of amyloid deposits was different among individual organs of single cases, especially in early-onset cases. As compared to early-onset cases, the characteristics of amyloid deposits in various organs of late-onset cases seemed to be rather uniform. Amyloid deposits in most organs in earlyonset cases were distinct from those in late-onset cases but those in the renal glomus and the adrenal gland in early-onset cases were rather similar to those in late-onset cases. In the present study we determined the proportion of variant TTR in total TTR of amyloid deposits in the heart only because we were able to extract sufficient amount of TTR for SELDI-TOF MS from this organ. As for the other organs, we obtained sufficient TTR from the sciatic nerve of patient 2. The percentage of variant TTR in total TTR in the sciatic nerve of this case was 71.6%, which was lower than that in the heart of the same case but still higher than those in the heart of the late-onset cases (Supplemental data). These observations may suggest that the environmental condition of the interstitial tissues of the individual organs, where amyloid deposits, determines the characteristics of amyloid deposits in each organ. For instance, extracellular matrix components including proteoglycans and glycosaminoglycans are known to be constituents of amyloid deposits [34], and these components may differ among respective organs, resulting in organ-specific timing of the initiation and the characteristics of amyloid deposition. A previous study has suggested the participation of extracellular matrix components, such as biglycan, neutrophil gelatinase-associated lipocalin, and matrix metalloproteinase-9 in FAP ATTR Val30Met patients [35]. It is known that the properties of extracellular matrix components change with aging [36], and they may determine the age at which amyloid deposition initiates. In earlyonset cases, some extrinsic or intrinsic factors, which may also be associated with anticipation of the age at onset, may change these components and result in the differential timing and nature of amyloid deposition. Identifying these factors may clarify the pathogenesis of organ-specific amyloid deposition in FAP ATTR Val30Met and the cause of the different phenotype in the early- and late-onset cases. In addition to liver transplantation, new approaches, such as the prevention of amyloid fibril formation by stabilizing TTR tetramers or the reduction of TTR deposition by immunization [37–39], have been developed as therapeutic strategies for FAP. Targeting those factors in the interstitial tissues that may be responsible for increased amyloid deposition may add new therapeutic strategies for the treatment of FAP. Since wild-type TTR may contribute considerably to amyloid deposition in the late-onset form of FAP, this concept may be additionally important. Likewise, it may also be possible to apply these therapeutic strategies to the treatment of SSA.
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Contents lists available at ScienceDirect
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
Distinct characteristics of amyloid deposits in early- and late-onset transthyretin Val30Met familial amyloid polyneuropathy Haruki Koike a, Yukio Ando b, Mitsuharu Ueda b, Yuichi Kawagashira a, Masahiro Iijima a, Junko Fujitake c, Michiyuki Hayashi c, Masahiko Yamamoto d, Eiichiro Mukai e, Tomohiko Nakamura a, Masahisa Katsuno a, Naoki Hattori a, Gen Sobue a,⁎ a
Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan Department of Diagnostic Medicine, Kumamoto University School of Medicine, Kumamoto, Japan c Department of Neurology, Kyoto City Hospital, Kyoto, Japan d Department of Speech Pathology and Audiology, Aichi Gakuin University School of Health Science, Aichi, Japan e Department of Neurology, Nagoya Medical Center, Nagoya, Japan b
a r t i c l e
i n f o
Article history: Received 25 March 2009 Rceived in revised form 29 July 2009 Accepted 31 July 2009 Available online 25 August 2009 Keywords: Familial amyloid polyneuropathy Amyloid Congo red staining Transthyretin Autopsy Surface enhanced desorption/ionization timeof-flight mass spectrometry
a b s t r a c t Late-onset transthyretin Val30Met-associated familial amyloid polyneuropathy (FAP ATTR Val30Met) cases unrelated to endemic foci in Japan show different clinicopathological features from the conventional earlyonset cases in endemic foci. We compared the characteristics of amyloid deposits in early-onset FAP ATTR Val30Met cases in endemic foci and late-onset cases in non-endemic areas. Amyloid deposits in three earlyonset cases from endemic foci and five late-onset cases from non-endemic areas were systematically examined post-mortem. Amyloid deposits in early-onset cases were highly congophilic and showed strong apple-green birefringence with Congo red staining and had long, parallel fibrils in most organs. On the other hand, those in late-onset cases were generally weakly congophilic and showed faint apple-green birefringence with Congo red staining and had short, haphazard fibrils. In the renal glomus and adrenal gland of early-onset cases, the characteristics of amyloid deposits were similar to those observed in lateonset cases. Analysis of cardiac amyloid using surface enhanced desorption/ionization time-of-flight mass spectrometry indicated that most transthyretin (TTR) was variant in early-onset cases, while more than half was composed of wild-type TTR in late-onset cases. Although characteristics of amyloid deposits may differ among individual organs of respective cases, especially in early-onset cases, the pattern was distinct between early- and late-onset cases. Amyloid deposition in late-onset cases may be similar to that observed in senile systemic amyloidosis with wild-type TTR deposition, suggesting that aging may play an important role in these cases. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Transthyretin Val30Met-associated familial amyloid polyneuropathy (FAP ATTR Val30Met) is the most common type of familial amyloid polyneuropathy [1–9]. The classical concept of this disease was that most patients have a relationship to endemic foci in Japan, Portugal, and Sweden [2–4,6–8]. However, through the development of gene diagnostic techniques, many patients unrelated to these endemic foci have been reported in many nations worldwide [5,9–12], suggesting that this disease is more prevalent than was generally believed. Previous studies of Japanese FAP ATTR Val30Met patients
⁎ Corresponding author. Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. Tel.: +81 52 744 2385; fax: +81 52 744 2384. E-mail address: [email protected] (G. Sobue). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.07.028
have demonstrated significant differences between patients in endemic foci and those in non-endemic areas [10,11,13,14]. Although there are exceptions, typical cases of FAP ATTR Val30Met in these endemic foci show characteristic features including an age at onset from the late 20 s to the early 40 s, a high penetrance rate, marked autonomic dysfunction, loss of superficial sensation including nociception and thermal sensation (i.e. sensory dissociation), atrioventricular conduction block requiring pacemaker implantation, and anticipation of the age at onset [2,3,8,11,15,16]. On the other hand, patients in non-endemic areas usually show an older age at onset over 50 years, a low penetrance rate, relatively mild autonomic dysfunction, loss of all sensory modalities rather than sensory dissociation, frequent presence of cardiomegaly, extreme male preponderance, and an absence of anticipation of the age at onset [10,11,13,14,16,17]. Differences in the geographical distribution and clinical features between the early- and late-onset types of FAP ATTR Val30Met have also been reported in Portugal [6,18,19]. The causal mechanism of the
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Table 1 Background of patients with early- and late-onset FAP ATTR Val30Met. Age at onset/ death (years)
Initial symptom
Sensory dissociation
Cardiac involvement Cardio-megalya
Pacemaker implantation
Early-onset casesb 1 F 2 F 3 M
28/35 37/51 24/41
A A A
+ + +
− − −
+ + −
Sudden death Pneumonia Pneumonia
Late-onset casesc 4 5 6 7 8
64/67 62/68 52/62 59/74 61/66
P P P P P
+ + − + −
− + + − +
− − − − −
Lung cancer Heart failure Heart failure Heart failure Heart failure
Patients
Sex
M M M M M
Cause of death
+ = present; − = absent; F = female; M = male; A = autonomic symptoms; P = paresthesia in the legs. a The presence of cardiomegaly was assessed at the time of first referral to the hospital. b Early-onset FAP ATTR Val30Met cases in endemic foci. c Late-onset FAP ATTR Val30Met cases in non-endemic areas.
differences between the early- and late-onset types of FAP with the same mutation in the TTR gene has not yet been determined. In this context, we systematically investigated and compared the characteristics of amyloid deposits in tissues from autopsied patients with conventional early-onset FAP ATTR Val30Met from endemic foci and late-onset FAP ATTR Val30Met unrelated to these endemic foci.
sections. All specimens from all cases were stained simultaneously for each method. For electron microscopy, formalin-fixed, paraffinembedded tissue sections were deparaffinized and embedded in epoxy resin. Epoxy-resin embedded specimens were cut into ultrathin sections and stained with uranyl acetate and lead citrate.
2. Materials and methods 2.1. Patients Eight autopsied cases with genetically-confirmed FAP ATTR Val30Met were examined: three early-onset patients (1 male and 2 females) from endemic foci; and five late-onset patients (5 males) from non-endemic areas of Japan. No patient in the study belonged to the same kindred as another. Informed consent was obtained from the patient's families, and all aspects of the study were approved by the Ethics Committees of Nagoya University Graduate School of Medicine. The characteristics of the patients are summarized in Table 1. Although the duration of the disease was longer in the early-onset cases (12.7 ± 5.1 years for early-onset cases and 7.8 ± 4.8 years for late-onset cases, mean ± SD), heart weight was heavier in the lateonset cases (437 ± 15 g in early-onset cases and 611 ± 160 g in lateonset cases). In the late-onset cases, the enlargement of the whole heart was evident and ventricular walls were markedly hypertrophic, except for case 4 whose duration of the disease was shortened due to lung cancer. Therefore, the cause of death in the late-onset cases was mainly due to heart failure. The pathological findings, focusing on the distribution of amyloid deposits in various organs in 7 of these cases (patients 1 to 7), have been reported previously [13,20]. 2.2. Pathological examinations The nervous system including the brain, spinal cord, ventral and dorsal roots, dorsal root ganglia from L3 to L5, sympathetic ganglia, proximal portion of the sciatic nerve, and visceral organs were removed, fixed in 10% formalin solution, and embedded in paraffin [13,21,22]. All specimens were cut into 4 μm thick sections by routine methods and stained with hematoxylin and eosin and alkaline Congo red. Congo red-stained sections were viewed under polarized light. The degree of the intensity for glittering birefringence after Congo red staining was quantified into three levels as follows: 1, low; 2, middle ; and 3, high. Immunohistochemical assessment was performed with a peroxidase-antiperoxidase method using an anti-human transthyretin (TTR) antibody (Dako; 1:1000) in consecutive deparaffinized
Table 2 Intensity of birefringence after Congo red staining and morphology of amyloid fibrils. Early-onset casesa (n = 3)
Late-onset casesb (n = 5) Congo redc
Morphologyd
Long, parallel
1.0
ND
ND
1.0
Thyroid gland
3.0
Long, parallel
1.6
Lung
3.0
Long, parallel
1.0
Gastrointestinal tract Spleen
3.0
Long, parallel
1.0
2.7
Long, parallel
1.0
Pancreas
3.0
Long, parallel
1.2
Adrenal gland
1.3
Short, haphazard
ND
Short, haphazard Short, haphazard Short, haphazard Short, haphazard Short, haphazard Short, haphazard Short, haphazard ND
Kidney Cortex
1.0
ND
ND
3.0
Short, haphazard Long, parallel
1.5
Short, haphazard
2.7
Long, parallel
1.3
Dorsal root ganglia 2.3
Long, parallel
1.0
Sympathetic ganglia
Long, parallel
1.0
Short, haphazard Short, haphazard Short, haphazard
Congo redc
Morphology
Heart
3.0
Hypophysis
Medulla Nervous system Sciatic nerve
2.3
d
ND = not determined due to little amyloid deposition. a Early-onset FAP ATTR Val30Met cases in endemic foci. b Late-onset FAP ATTR Val30Met cases in non-endemic areas. c The degree of the intensity for glittering birefringence after Congo red staining was quantified into three levels as follows: 1, low; 2, middle ; and 3, high. Values were expressed as the mean. d Morphology of amyloid fibrils was determined using electron microscopy.
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Fig. 1. Representative characteristics of amyloid deposits in the heart from early-onset (A to D) and late-onset cases (E to H). (A, B, E, and F) Alkaline Congo red staining. (C and G) Immunostaining with the anti-human transthyretin (TTR) antibody using consecutive sections for Congo red staining. (D and H) Morphology of amyloid fibrils as observed with electron microscopy. By Congo red staining, amyloid deposits in early-onset cases were highly congophilic (A) and had bright and glittering apple-green birefringence when examined by polarized light (B), while those in late-onset cases were weakly congophilic (E) and showed faint birefringence under polarized light (F). Affinity to the anti-TTR antibody was strong in early-onset cases (C), while it was weak in late-onset cases (G). By electron microscopy, straight long fibrils that were often aligned in a parallel fashion were observed in early-onset cases (D), while fairly short and haphazardly arranged fibrils were present in late-onset cases (H). Bar = 0.3 μm.
2.3. Analysis of TTR in amyloid deposits To determine the proportion of variant (Val30Met) TTR in total (Val30Met plus wild-type) TTR in amyloid deposits, we used surface enhanced desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Amyloid deposits in the hearts from three earlyonset cases (patients 1 to 3) and four late-onset cases (patients 4 to 7) were analyzed. Tissue-deposited TTR was extracted in 6 M guanidine hydrochloride in 0.15 M Tris-HCl buffer, pH 8.0, from formalin-fixed paraffin sections as described previously [23]. Samples were purified by means of reversed-phase HPLC (Waters 600E HPLC system, Waters Co.) on an Aquapore C8 column (7 mm, 300 Å, 30× 4.6 mm, PerkinElmer), as described previously [23]. The collected TTR-rich fractions were dialyzed in 50 mM phosphate buffer, pH 6.0, and were used for SELDITOF-MS analyses. For SELDI-TOF MS, a strong anion exchange array with quaternary amine functionality (Q10) was used. Binding of proteins to ProteinChip surfaces was performed in a 96-well format bioprocessor (Bio-Rad Laboratories). Samples were analyzed with the PCS 4000 SELDI-TOF-MS instrument (Bio-Rad Laboratories). ProteinChips were read by using the following protocol: ion focus mass, 13,800 m/z; laser energy, 2000 nJ; matrix attenuation, 500 m/z; sample rate, 800; shots/pixel, 5; partition, 1 of 4; and acquired mass range from 0 to 100,000 m/z. External calibration of the instrument was performed by using the All-in-1 protein molecular mass standard (Bio-Rad Laboratories). Using the regression equation obtained in the quanti-
tative analysis of the ratio of variant to wild-type TTR via Q10 ProteinChip, we calculated the percentage of variant TTR in total TTR in amyloid deposits. To determine the peaks of variant TTR and wildtype TTR, sera from a normal subject and a heterozygous FAP ATTR Val30Met patient were also analyzed. 3. Results 3.1. Characteristics of amyloid deposits The characteristics of amyloid deposits in various organs are summarized in Table 2. As previously described [13], amyloid deposition was prominent in the heart, thyroid gland, gastrointestinal tract, pancreas, adrenal gland, kidney, and peripheral nervous system in early-onset cases. In late-onset cases, although the amount of amyloid deposits was generally less than that observed in early-onset cases, it was more conspicuous in the heart and the anterior lobe of the pituitary gland as compared to early-onset cases [13,20]. Because amyloid deposition was scarce, limited around small vessels, or absent in the parenchyma of the central nervous system, the posterior lobe of the pituitary gland, liver, and skeletal muscle in both groups, we did not assess the characteristics of amyloid deposits in these organs. When the sections were stained with alkaline Congo red, amyloid deposits in early-onset cases were highly congophilic and had bright and glittering apple-green birefringence when examined by polarized light (Table 2; Fig. 1A and B), except for those in the renal glomus and
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Fig. 2. Representative characteristics of amyloid deposits in the kidney from early-onset cases. (A and B) Alkaline Congo red staining. (C) Immunostaining with the anti-human transthyretin (TTR) antibody using consecutive sections for Congo red staining. (D and E) Morphology of amyloid fibrils as observed with electron microscopy. In A to C, the renal glomus is observed on the upper side of the photograph, while the medulla is located on the lower side. In the medulla, the affinity of amyloid deposits to Congo red was high (A, lower side) and showed bright apple-green birefringence (B, lower side), while it was weak (A, upper side) and showed faint birefringence (B, upper side) in the glomus. Affinity to the anti-TTR antibody was strong in the medulla (C, lower side), while it was weak in the glomus (C, upper side). By electron microscopy, straight long fibrils that were often aligned in a parallel fashion were observed in the medulla (D), while fairly short and haphazardly arranged fibrils were observed in the glomus (E). Bar = 0.3 μm.
adrenal gland. In the kidneys of early-onset cases, the affinity to Congo red of amyloid deposits was high and showed bright apple-green birefringence in the medulla, while affinity to Congo red of amyloid in the glomus was weak and showed faint birefringence (Fig. 2A and B). Interestingly, in the renal cortex, the affinity of amyloid to Congo red in the interstitium or vessel wall was high, showing a similar pattern to the other organs, despite its close proximity to the glomerular amyloid. Amyloid deposits in the adrenal gland also showed similar characteristics to those in the renal glomus. On the other hand, the amyloid deposits in the organs from late-onset cases were generally weakly congophilic and showed faint birefringence under polarized light as compared to those in most organs from the early-onset cases (Table 2; Fig. 1E and F). The intensity of TTR immunostaining of amyloid deposits showed similar tendency to the affinity to Congo red; it was largely higher in the early-onset cases than in the lateonset cases in most organs (Fig. 1C and G). Amyloid deposits in the renal glomus and adrenal gland of early-onset cases tended to show weak affinity to the anti-TTR antibody, which is similar to that observed in the late-onset cases (Fig. 2C). Electron microscopy revealed two patterns of amyloid fibrils corresponding well to the affinity to Congo red (Table 2). In amyloid deposits with a high affinity to Congo red seen in most organs in earlyonset cases, straight long fibrils that were often aligned in a parallel fashion were observed (Figs. 1D and 2D). On the other hand, fairly short and haphazardly arranged fibrils were present in the amyloid deposits with a low affinity to Congo red that were seen in the organs from late-onset cases and in the renal glomus and adrenal gland in early-onset cases (Figs. 1H and 2E). 3.2. The ratio of variant to wild-type TTR in amyloid deposits Fig. 3 shows SELDI-TOF mass spectra of TTR prepared from cardiac amyloid in early- and late-onset cases. A peak in early-onset cases showed a molecular mass of 13,792 m/z, corresponding to the variant TTR. On the other hand, two peaks were observed in the late-onset cases; one of which showed a molecular mass of 13,792 m/z,
corresponding to the variant TTR, and another showed 13,761 m/z, corresponding to the wild-type TTR, which was not detected in the early-onset cases. The percentage of variant TTR in total TTR in amyloid deposits was seen to be 42.3%, 41.4%, 37.1%, and 44.0% in patients 4 to 7, respectively. Because the peak of wild-type TTR was not detected in any early-onset cases, we considered the percentage of variant TTR in these cases to be more than 80%. 4. Discussion In this study, we examined the characteristics of amyloid deposits in patients with FAP ATTR Val30Met in Japan. By comparing the characteristics of Congo red staining and morphology of amyloid fibrils in conventional early-onset cases from endemic foci to those in late-onset cases from non-endemic areas, we demonstrated clear differences of amyloid deposits between these two types of FAP ATTR Val30Met. Amyloid deposits in early-onset cases tended to be highly congophilic and showed strong apple-green birefringence in Congo red staining and had long, parallel fibrils in most organs. On the other hand, those in late-onset cases were generally weakly congophilic and showed faint apple-green birefringence in Congo red staining and had short, haphazard fibrils. The intensity of TTR immunostaining also was different between early- and late-onset cases; it was largely higher in the early-onset cases than in the late-onset cases in most organs. We have to keep in mind the reproducibility of staining properties of these methods with caution. To minimize the ambiguity, we stained all sections simultaneously for each method. Also, we admit formalinfixed samples to be less suitable than glutaraldehyde-fixed samples for electron microscopic examination. However, by using formalinfixed samples, we were able to examine many sections of various organs. It enabled us to discover an entire spectrum of the morphology of amyloid fibrils. To confirm the difference between early- and late-onset cases, we used SELDI-TOF MS, which showed distinct characteristics of amyloid deposits. Deposited TTR in the heart was mostly variant in early-onset cases, while abundant wild-type TTR contributed in late-onset cases.
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Fig. 3. Mass spectra of transthyretin (TTR) prepared from cardiac amyloid in early-onset cases (patients 1 to 3) and late-onset cases (patients 4 to 7) by surface enhanced desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF MS). To determine the peaks of variant (Val30Met) TTR and wild-type TTR, sera from a normal subject and a heterozygous FAP ATTR Val30Met patient were also analyzed. A peak in early-onset cases showed a molecular mass of 13,792 m/z (arrowheads), corresponding to the variant TTR. On the other hand, two peaks were observed in late-onset cases; one of which showed a molecular mass of 13,792 m/z (arrowheads), corresponding to the variant TTR, and another showed 13,761 m/z (asterisks), corresponding to the wild-type TTR, which was not detected in early-onset cases. The percentage of variant TTR in total TTR in amyloid deposition was seen to be 42.3%, 41.4%, 37.1%, and 44.0% in patients 4 to 7, respectively. Because the peak of wild-type TTR was not detected in any of the early-onset cases, we considered the percentage of variant TTR in these cases to be more than 80%.
This abundance of wild-type TTR in late-onset cases may support the view that the mechanism of amyloid deposition is similar, to some extent, to that observed in senile systemic amyloidosis (SSA). In SSA, wild-type TTR is known to be an amyloid precursor protein [24]. SSA predominantly affects men [25,26]. The presence of an extreme male preponderance in late-onset FAP ATTR Val30Met in non-endemic areas, as opposed to a nearly 1 to 1 male–female ratio in early-onset cases in endemic foci, is also similar to that observed in SSA [10,11,26]. In addition, amyloid deposition in the heart and anterior pituitary gland, that characterized the late-onset FAP ATTR Val30Met cases in non-endemic areas, is sometimes found in the normal elderly population [27–30]. These findings suggest the importance of unidentified factors that change with aging to the amyloid deposition in late-onset FAP ATTR Val30Met cases. A previous study of cardiac amyloid deposits in Swedish patients with FAP ATTR Val30Met and SSA have demonstrated two distinct
patterns of amyloid deposition; one pattern is similar to the amyloid deposits from our early-onset cases, while the other pattern resembles those from our late-onset cases [31]. Swedish FAP ATTR Val30Met patients showed the former pattern in 6 of 11 patients and the latter pattern in 5 of 11 patients, while all SSA cases showed the latter pattern [31]. A recent study analyzing amyloid deposits in subcutaneous adipose tissue from Swedish FAP ATTR Val30Met patients revealed that the former pattern tended to be present in patients with an early-age at onset, while the latter pattern was detected in relatively late-age at onset patients [32]. The features of FAP ATTR Val30Met in a Swedish endemic focus are not similar to those in Japanese and Portuguese endemic foci but rather resemble those in late-onset cases from non-endemic areas of Japan i.e. the mean age at onset in the fifth decade of life, a low penetrance rate, a tendency to manifest sensori-motor neuropathy rather than autonomic symptoms as an initial symptom, and extensive amyloid
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deposition in the heart [1,4]. In some Swedish FAP ATTR Val30Met patients, symptoms of cardiac failure are more prominent than those of neuropathy [32]. The age at onset in these patients tend to be elderly and amyloid deposits in such patients show characteristics similar to those in SSA [31,32]. In addition, a recent epidemiological study showed the presence of a new endemic focus (Ishikawa prefecture) outside the conventional endemic foci (Arao and Ogawa) in Japan [33]. Features of patients in this new focus are different from those in the conventional endemic foci, but resemble those in Sweden [33]. Therefore, FAP ATTR Val30Met patients in Japan could now be classified into: (1) conventional early-onset cases in endemic foci (Arao and Ogawa); (2) late-onset cases in an endemic focus (Ishikawa); and (3) late-onset cases in non-endemic areas. In this context, cases in a Swedish endemic focus and Ishikawa prefecture seem to be an intermediate form between cases in nonendemic areas and those in other endemic foci. Because the late-onset type of FAP ATTR Val30Met in non-endemic areas is considered to be more prevalent in Japan and elsewhere than was generally appreciated [11,14], it may be hypothesized that it is a prototype of FAP ATTR Val30Met. Some indigenous environmental or genetic factors may have been created endemic foci and some of them induced anticipation creating the early-onset type of FAP ATTR Val30Met. Another interesting finding is that the characteristic of amyloid deposits was different among individual organs of single cases, especially in early-onset cases. As compared to early-onset cases, the characteristics of amyloid deposits in various organs of late-onset cases seemed to be rather uniform. Amyloid deposits in most organs in earlyonset cases were distinct from those in late-onset cases but those in the renal glomus and the adrenal gland in early-onset cases were rather similar to those in late-onset cases. In the present study we determined the proportion of variant TTR in total TTR of amyloid deposits in the heart only because we were able to extract sufficient amount of TTR for SELDI-TOF MS from this organ. As for the other organs, we obtained sufficient TTR from the sciatic nerve of patient 2. The percentage of variant TTR in total TTR in the sciatic nerve of this case was 71.6%, which was lower than that in the heart of the same case but still higher than those in the heart of the late-onset cases (Supplemental data). These observations may suggest that the environmental condition of the interstitial tissues of the individual organs, where amyloid deposits, determines the characteristics of amyloid deposits in each organ. For instance, extracellular matrix components including proteoglycans and glycosaminoglycans are known to be constituents of amyloid deposits [34], and these components may differ among respective organs, resulting in organ-specific timing of the initiation and the characteristics of amyloid deposition. A previous study has suggested the participation of extracellular matrix components, such as biglycan, neutrophil gelatinase-associated lipocalin, and matrix metalloproteinase-9 in FAP ATTR Val30Met patients [35]. It is known that the properties of extracellular matrix components change with aging [36], and they may determine the age at which amyloid deposition initiates. In earlyonset cases, some extrinsic or intrinsic factors, which may also be associated with anticipation of the age at onset, may change these components and result in the differential timing and nature of amyloid deposition. Identifying these factors may clarify the pathogenesis of organ-specific amyloid deposition in FAP ATTR Val30Met and the cause of the different phenotype in the early- and late-onset cases. In addition to liver transplantation, new approaches, such as the prevention of amyloid fibril formation by stabilizing TTR tetramers or the reduction of TTR deposition by immunization [37–39], have been developed as therapeutic strategies for FAP. Targeting those factors in the interstitial tissues that may be responsible for increased amyloid deposition may add new therapeutic strategies for the treatment of FAP. Since wild-type TTR may contribute considerably to amyloid deposition in the late-onset form of FAP, this concept may be additionally important. Likewise, it may also be possible to apply these therapeutic strategies to the treatment of SSA.
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