Toshinori Oshima

Clinicopathological features of senile systemic amyloidosis: an ante- and post-mortem study

Senile systemic amyloidosis is a common age-related amyloidosis that involves accumulation of wild-type transthyretin, with cardiac dysfunction being a predominant result. The importance of obtaining an accurate diagnosis of senile systemic amyloidosis has been increasingly recognized, so that novel treatments are being developed. However, the clinicopathological features of senile systemic amyloidosis remain to be completely understood. Here, we evaluated cardiac specimens from 181 consecutive post-mortem cases older than 40 years, including 6 cases of senile systemic amyloidosis, and 5 cases of familial amyloidotic polyneuropathy, which is a hereditary systemic amyloidosis caused by mutant forms of transthyretin. Furthermore, we studied ante-mortem clinicopathological findings of 11 senile systemic amyloidosis cases, in which 9 cases underwent gastrointestinal tract biopsy and/or subcutaneous tissue biopsy, at Kumamoto University Hospital. Of the autopsied cases of elderly Japanese (older than 80 years), 12% had senile systemic amyloidosis, with the percentage increasing with age. The occurrence of senile systemic amyloidosis in elderly Japanese patients was lower than that in previous reports, which suggests that a genetic background and/or environmental factor(s) may have important roles in the occurrence of senile systemic amyloidosis. Transthyretin amyloid deposits in familial amyloidotic polyneuropathy cases developed mainly in the pericardium and the surrounding muscle fascicles, whereas in cases with senile systemic amyloidosis the transthyretin amyloid deposits had a patchy plaque-like shape and developed mainly inside the ventricular wall. Biopsies from senile systemic amyloidosis patients evidenced amyloid deposits in 44% (4/9) of gastrointestinal tract and subcutaneous tissue samples combined. As myocardial biopsy may be dangerous for elderly people, the use of a combination of gastrointestinal tract and subcutaneous tissue biopsies may make diagnosis of senile systemic amyloidosis easier.

Changes in pathological and biochemical findings of systemic tissue sites in familial amyloid polyneuropathy more than 10 years after liver transplantation

Objective To elucidate the long-term effects of liver transplantation (LT) on familial amyloid polyneuropathy (FAP). Methods We investigated clinicopathological and biochemical characteristics of systemic tissues in four autopsied cases of FAP patients surviving more than 10 years after LT and seven autopsied cases without LT. For analysing the truncated form of transthyretin (TTR) in amyloid, we also employed specimens from additional 18 FAP patients. Results Several tissue sites such as the heart, tongue and spinal cord had moderate-to-severe amyloid deposits but other tissues showed no or mild amyloid deposition. Those findings seemed similar to those observed in senile systemic amyloidosis (SSA), a sporadic amyloidosis caused by wild-type (WT) TTR. Also, amyloid deposits in systemic tissue sites except for the spinal cord in patients after LT derived mostly from WT TTR secreted from the normal liver grafts. In addition, in non-transplantation patients, proportions of WT TTR seemed to be relatively high in those tissue sites in which patients after LT had severe amyloid deposition, which suggests that WT TTR tends to form amyloid in those tissue sites. Finally, although the truncation of TTR in amyloid deposits did not depend on undergoing LT, we elucidated the truncation of TTR occurred predominantly in patients from non-endemic areas of Japan, where FAP amyloidogenic TTR V30M patients are late onset and low penetrance, compared with patients from an endemic area of Japan. Conclusions FAP may shift to systemic WT TTR amyloid formation after LT, which seems to be similar to the process in SSA. The truncation of TTR in amyloid deposits may depend on some genetic or environmental factors other than undergoing LT.

Effect of age and sex differences on wild-type transthyretin amyloid formation in familial amyloidotic polyneuropathy: A proteomic approach

BACKGROUND Age and sex differences are closely related to the onset of senile systemic amyloidosis (SSA) caused by wild-type (WT) transthyretin (TTR). However, the effects of these differences on the amyloid formation mechanism in familial amyloid polyneuropathy (FAP) caused by variant TTR, have remained unclear. To elucidate age and sex differences in FAP, we investigated biochemical characteristics of amyloid deposits in different tissue sites of FAP by proteomic analysis. METHODS We used shotgun liquid chromatography/tandem mass spectrometry to analyze the proportions of variant and WT TTR in amyloid deposits in different tissues, such as cardiac, kidney, peripheral nerves, and gastrointestinal tissues, from 23 autopsied FAP cases. RESULTS AND CONCLUSIONS The analysis revealed a highly significant correlation between the proportion of WT TTR and age at autopsy in cardiac tissues, whereas the analysis indicated no correlation in kidney, peripheral nerves, and gastrointestinal tissues. In addition, we demonstrated age-related significantly increased WT TTR deposits, but not variant TTR deposits, in cardiac tissues of male patients. Taken together, these data suggest that both age and sex differences affect cardiac amyloid formation, mainly derived from WT TTR, in FAP.

Amyloid deposits derived from transthyretin in the ligamentum flavum as related to lumbar spinal canal stenosis

Amyloidosis is a protein conformational disorder with the distinctive feature of extracellular accumulation of amyloid fibrils that come from different proteins. In the ligamentum flavum of the lumbar spine, amyloid deposits were frequently found in elderly patients with lumbar spinal canal stenosis and were at least partially formed by wild-type transthyretin. However, how amyloid deposits in the ligamentum flavum affect lumbar spinal canal stenosis has remained unclear. In this study, we analyzed clinical, pathologic, and radiologic findings of patients with lumbar spinal canal stenosis who had amyloid deposits in the ligamentum flavum. We studied 95 ligamentum flavum specimens obtained from 56 patients with lumbar spinal canal stenosis and 21 ligamentum flavum specimens obtained from 19 patients with lumbar disk herniation. We evaluated histopathologic findings and clinicoradiologic manifestations, such as thickness of the ligamentum flavum and lumbar spinal segmental instability. We found that all 95 ligamentum flavum specimens resected from patients with lumbar spinal canal stenosis had amyloid deposits, which we classified into two types, transthyretin-positive and transthyretin-negative, and that transthyretin amyloid formation in the ligamentum flavum of patients with lumbar spinal canal stenosis was an age-associated phenomenon. The amount of amyloid in the ligamentum flavum was related to clinical manifestations of lumbar spinal canal stenosis, such as thickness of the ligamentum flavum and lumbar spinal segmental instability, in the patients with lumbar spinal canal stenosis with transthyretin-positive amyloid deposits. To our knowledge, this report is the first to show clinicopathologic correlations in transthyretin amyloid deposits of the ligamentum flavum. In conclusion, transthyretin amyloid deposits in the ligamentum flavum may be related to the pathogenesis of lumbar spinal canal stenosis in elderly patients.

Cyclodextrin, a novel therapeutic tool for suppressing amyloidogenic transthyretin misfolding in transthyretin-related amyloidosis

TTR (transthyretin), a β-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched β-CyDs, GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-β-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-β-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.

Spinal multifocal amyloidosis derived from wild-type transthyretin

Abstract Spinal amyloidosis can occur as a part of systemic amyloidosis or as localized amyloidomas. However, the exact pathogenesis of the spinal amyloidosis remains to be fully understood. Transthyretin (TTR) is an amyloidogenic protein causing two kinds of amyloid diseases. One is senile systemic amyloidosis (SSA), which is caused by wild-type (WT) TTR and primarily affects cardiac functions. The other type is familial amyloidosis, which is mainly induced by mutated TTR. We report here the first case of multifocal spinal TTR amyloidosis derived from WT TTR with radiculomyelopathy and destructive spondylosis. The data and clinical manifestations suggest that the patient may develop SSA. Clinical manifestations of TTR-related amyloidosis may vary more than we previously thought. In spinal amyloidosis, WT TTR is one of the candidate precursor proteins for the disease.

Recipient aging accelerates acquired transthyretin amyloidosis after domino liver transplantation

Domino liver transplantation (DLT) with liver grafts from patients with hereditary transthyretin (TTR) amyloidosis has been performed throughout the world because of a severe liver graft shortage. Reports of acquired systemic TTR amyloidosis in domino liver recipients have been increasing; however, the precise pathogenesis and clinical course of acquired TTR amyloidosis remains unclear. We analyzed the relationship between the occurrence of acquired amyloidosis and clinical features in 22 consecutive domino liver donors with hereditary TTR amyloidosis (10 males and 12 females; mean age at DLT: 37.2 years; TTR mutations: V30M [n = 19], Y114C [n = 1], L55P [n = 1], and S50I [n = 1]) and 22 liver recipients (16 males and 6 females; mean age at DLT, 46.2 years). The mean times from DLT to amyloid first appearance and transplant recipient symptom onset were 8.2 years and 9.9 years, respectively. Kaplan‐Meier analysis and quantification of the amyloid deposition revealed aging of recipients correlated with early de novo amyloid deposition. The sex of donors and recipients and the age, disease duration, and disease severity of donors had no significant effect on the latency of de novo amyloid deposition. In conclusion, our results demonstrate that recipient aging is associated with the early onset de novo amyloidosis. Because acquired amyloidosis will likely increase, careful follow‐up for early amyloidosis detection and new treatments, including TTR stabilizers and gene‐silencing therapies, are required. Liver Transplantation 22 656‐664 2016 AASLD.

Pathological changes long after liver transplantation in a familial amyloidotic polyneuropathy patient

Liver transplantation (LT) reportedly prolongs the survival of patients with familial amyloidotic polyneuropathy (FAP), a fatal hereditary systemic amyloidosis caused by mutant transthyretin (TTR). However, what happens in systemic tissue sites long after LT is poorly understood. In the present study, we report pathological and biochemical findings for an FAP patient who underwent LT and died from refractory ventricular fibrillation more than 16 years after FAP onset. Our autopsy study revealed that the distributions of amyloid deposits after LT were quite different from those in FAP amyloidogenic TTR V30M patients not having had LT and seemed to be similar to those observed in senile systemic amyloidosis (SSA), a sporadic systemic amyloidosis derived from wild-type (WT) TTR. Our biochemical examination also revealed that this patients cardiac and tongue amyloid deposits derived mostly from WT TTR. We propose that FAP patients after LT may suffer from SSA-like WT TTR amyloidosis in systemic organs.

Impact of antibodies against amyloidogenic transthyretin (ATTR) on phenotypes of patients with familial amyloidotic polyneuropathy (FAP) ATTR Valine30Methionine.

BACKGROUND This study investigated whether a relationship exists between the presence of de novo antibodies and the clinical manifestations of familial amyloidotic polyneuropathy (FAP). METHODS Serum samples were collected from 25 Japanese and 6 Swedish FAP amyloidogenic transthyretin (ATTR) Valine30Methionine (V30M) patients, 4 asymptomatic Japanese ATTR V30M gene carriers, and 24 Japanese healthy volunteers. Study methods included enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. RESULTS Three Japanese and 5 Swedish patients had significantly higher levels of antibodies against ATTR than did healthy volunteers and asymptomatic gene carriers (P<0.05). All 8 patients with higher antibody levels were late-onset cases. The ratio of wild-type TTR to ATTR V30M in serum from the high-antibody group was higher than that of the low-antibody group. ELISA results revealed two epitopes at positions 24-35 and 105-115 of ATTR V30M. We found a significant positive correlation between levels of the antibody at positions 24-35 and the age at FAP onset (r=0.751, P<0.05). An age-dependent increase in the occurrence of antibodies was observed in these patients with an epitope at positions 24-35. CONCLUSIONS These findings may help explain the differences in early- and late-onset FAP and/or the progression of FAP.

A homozygote case of familial amyloid polyneuropathy amyloidgenic transthyretin Val30Met in a non-endemic area.

Homozygotic cases of familial amyloid polyneuropathy (FAP) are expected to show late onset and mild clinical manifestations, although a homozygote case of FAP showing extremely early onset and severe manifestations was reported recently. Clinical aspects of homozygotic cases of FAP remain controversial. We report a clinical feature of a homozygotic case of FAP in a non-endemic area. The case presented late onset, slow progression, an initial symptom of visual loss, small fiber neuropathy, and autonomic dysfunction. Serum total transthyretin (TTR) levels were relatively higher than those of heterozygotic cases of FAP. Homozygotic combination of stable tetramer of ATTR may have contributed to late onset and slow progression in this case. Introduction: Familial amyloid polyneuropathy (FAP), a fatal inherited autosomal dominant disorder, is characterized by systemic accumulation of polymerized amyloidogenic transthyretin (ATTR) in the peripheral nerves and systemic organs [1,2]. It has recently been suggested that instability of heterozygotic tetramer of variant transthyretin (TTR) structures causes dissociation of the tetramer and amyloid fibril formation [3–5]. It was reported that homozygotic tetramer of TTR was more stable than heterozygotic. Homozygotic cases of FAP are expected to show late onset and mild clinical manifestations [6– 8]. However, a homozygote case of FAP showing extremely early onset and severe manifestations was reported recently [9]. Clinical aspects of homozygotic cases of FAP remain controversial. We report clinical features of a homozygotic case of familial FAP ATTR Val30Met in a non-endemic area. Case report: A 72-year old Japanese female living in Hiroshima was admitted to Kumamoto University Hospital in June 2009 suffering from bilateral vitreous opacities and polyneuropathy. She suffered from paroxysmal atrial fibrillation at the age of 62 and cystitis at the age of 67. In her familial history, her aunt had complete AV block at the age of 70 and dysesthesia in her lower extremities at the age of 85. Her parents are cousins and did not have any relations in an endemic area of FAP foci. The patient had slow progressive visual loss at the age of 58. She developed vitreous opacities at the age of 59 but dysesthesia only at the age of 68. Upon admission, physical examination revealed normal modified body mass index (mBMI) (836 kg g/l m), mild orthostatic hypotension, and edema in left lower leg. Neurological examinations revealed visual loss, pupil border irregularity, dry eye, distal dominant muscle atrophy, and weakness in her lower extremities, absent Achilles’ tendon reflexes, dysesthesia and decreased pain and light touch sensation under knees and elbow with dissociated sensory loss, alternating constipation and diarrhea, and stress urinary incontinence. Total serum TTR were 21.8 mg/dl; relatively higher than those usually found in FAP ATTR Val30Met patients. The case did not present hypothyroidism. Nerve conduction studies showed normal motor nerve conduction velocity, decreased compound muscle action potential (CMAP), and absent sensory nerve action potential (SNAP) in the left ulnar nerve. CMAPs and SNAPs in the lower limbs were not observed. Holter ECG showed PACs and PVCs. R-R interval analysis revealed decreased CVRR (1.44, normal values42.81). Echocardiography did not show either granular sparkling sign, thickening of the intra ventricular septum, nor dilatation of left atrial diameter. Abdominal echography did not show reverse liver–kidney contrast indicating no renal involvement [1–3]. I-MIBG myocardial scintigraphy showed normal accumulation in the heart (early H/M ratio: 3.38, delayed H/M ratio1⁄4 3.13, washout rate 37.3%). Laser Doppler skin blood flow showed vascular hyper reactivity after deep breathing in especially lower extremities. On ophthalmological examination, visual acuities were decreased (0.05 and 0.02) and ocular tensions were normal (5 mmHg and 16 mmHg). Schirmer’s test (3 mm) revealed decreased tear production. Irregularity and amyloid deposition were observed on the pupillay margins. Funduscopic analysis revealed vitreous opacities caused by amyloid deposition. Histopathological examination revealed slight amyloid deposition on the gastric mucosa with Congo red staining under polarized light, although amyloid deposition was not detected in the duodenum mucosa or abdominal fat. After confirming the immunoreactivity of TTR in the amyloid deposits of gastric mucosa using an anti-TTR polyclonal antibody, surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI TOFMS) was used. A single peak corresponding to ATTR Val30Met was detected in the serum. Direct sequence analysis confirmed the homozygotic mutation in the TTR gene presenting one base substitution at codon 30 from GTG (Val) to ATG (Met). Discussion: We report a clinical feature of a homozygotic case of FAP in a non-endemic area. This case showed late onset, slow progression, an initial symptom of visual loss, small fiber neuropathy, 169