M. Ueda

Long-term survival after liver transplantation in patients with familial amyloid polyneuropathy

Objective: Familial amyloid polyneuropathy (FAP), which is a fatal disorder inherited in an autosomal dominant fashion, is characterized by systemic accumulation of polymerized transthyretin (TTR) in the peripheral nerves and systemic organs. Liver transplantation has become an accepted treatment of this disorder because it stops the major production of amyloidogenic TTR. However, improved survival of transplant patients compared with that of nontransplant patients has not been sufficiently demonstrated. This study investigated whether transplantation improved the long-term outcome of patients by comparing the survival of patients who had transplantations with that of patients who had not had transplantations. Methods: Eighty consecutive patients with FAP Val30Met who visited Kumamoto University Hospital between January 1990 and December 2010 were studied. The transplant group consisted of 37 patients who had a partial hepatic graft via living donor transplantation in Japan or who underwent liver transplantation in Sweden, Australia, or the United States. The nontransplant group consisted of 43 patients with FAP. Survival was evaluated by using Kaplan-Meier analysis, and the difference in survival was examined via the log-rank test. Results: The transplant group had prolonged survival (p < 0.001) compared with the nontransplant group. The estimated probability of survival at 10 years was 56.1% for the nontransplant group vs 100% for the transplant group. Conclusion: Liver transplantation should be considered as an effective treatment in clinical management of patients with FAP Val30Met. Classification of evidence: This study provides Class III evidence that liver transplantation prolongs survival in patients with FAP Val30Met.

Iatrogenic Amyloid Neuropathy in a Japanese Patient After Sequential Liver Transplantation

A 57‐year‐old woman in Japan, the first recipient of part of a liver from a 58‐year‐old man with familial amyloidotic polyneuropathy (FAP) amyloidogenic transthyretin Val30Met who had had sensorimotor polyneuropathy in the lower limbs for 3 years, started to develop sensory neuropathy 7 years after transplantation. Before the July 1998 sequential transplantation, she had been in a hepatic coma at the terminal stage of primary biliary cirrhosis and waiting for deceased donor liver transplantation. In September 2004, biopsy samples of her duodenum first showed amyloid deposition. Although biopsy materials in 2005 and 2006 showed no changes in amyloid deposition, decreased temperature sensation and pain in fingertips and toes were detected at a neurologic examination in March 2006. Thus, clinical symptoms of FAP appeared about 2 years after amyloid deposition started. Nerve conduction velocity studies revealed mild to moderate axonal sensory polyneuropathy without demyelination. Our findings confirmed iatrogenic sensory neuropathy induced by amyloid deposition 7 years after sequential liver transplantation.

Amyloid fibrils containing fragmented ATTR may be the standard fibril composition in ATTR amyloidosis

Abstract The clinical phenotype of familial ATTR amyloidosis depends to some extent on the particular mutation, but differences exist also within mutations. We have previously described that two types of amyloid fibril compositions exist among Swedish ATTRV30M amyloidosis patients, one consisting of a mixture of intact and fragmented ATTR (type A) and one consisting of mainly intact ATTR (type B). The fibril types are correlated to phenotypic differences. Patients with ATTR fragments have a late onset and develop cardiomyopathy, while patients without fragments have an early onset and less myocardial involvement. The present study aimed to determine whether this correlation between fibril type and phenotype is valid for familial ATTR amyloidosis in general. Cardiac or adipose tissues from 63 patients carrying 29 different TTR non-V30M mutations as well as 13 Japanese ATTRV30M patients were examined. Fibril type was determined by western blotting and compared to the patients’ age of onset and degree of cardiomyopathy. All ATTR non-V30M patients had a fibril composition with ATTR fragments, except two ATTRY114C patients. No clear conclusions could be drawn about a phenotype to fibril type correlation among ATTR non-V30M patients. In contrast, Japanese ATTRV30M patients showed a similar correlation as previously described for Swedish ATTRV30M patients. This study shows that a fibril composition with fragmented ATTR is very common in ATTR amyloidosis, and suggests that fibrils composed of only full-length ATTR is an exception found only in a subset of patients.

Neuroradiologic and clinicopathologic features of oculoleptomeningeal type amyloidosis

Objective: To clarify the pathogenesis of leptomeningeal amyloidosis in familial amyloidotic polyneuropathy amyloidogenic transthyretin Y114C (FAP ATTR Y114C). Methods: The authors analyzed eight FAP ATTR Y114C patients. Six patients showed CNS symptoms associated with leptomeningeal amyloidosis. To examine the function of the blood–CSF barrier and blood–brain barrier (BBB), the authors performed CSF and MRI studies. The authors also performed a histopathologic study of autopsy specimens to examine the distribution of amyloid deposition in the CNS. Results: CSF study showed high total protein concentrations and increased albumin CSF/serum concentration quotients (Qalb; an indication of blood–CSF barrier function). MRI with gadolinium (Gd) revealed enhancement from brainstem to spinal cord. Serial brain MRI studies with FLAIR images after Gd administration showed Gd leakage into the subarachnoid space (two patients). These findings suggested the blood–CSF barrier and BBB dysfunctions. Constructive interference in steady state (CISS) three-dimensional Fourier transformation (CISS-3DFT) sequence analysis demonstrated amyloid-induced funiculus structures joining the spinal cord and dura mater (one patient). Histopathologic study revealed intense amyloid deposition in leptomeninges, vessel walls, and parenchyma in spinal cord and the brain. These distributions of amyloid deposition are unique compared to other TTR related leptomeningeal amyloidosis. Conclusions: Patients with familial amyloidotic polyneuropathy amyloidogenic transthyretin Y114C had CNS disorders related to amyloid deposition in leptomeninges, vessel walls, and parenchyma in spinal cord and the brain. The pathogenesis of CNS disorders may reflect disruption of the blood–CSF barrier and blood–brain barrier by amyloid deposition.

Lactoferrin Glu561Asp facilitates secondary amyloidosis in the cornea

Aim: To elucidate the pathogenic mechanism of amyloid formation in corneal amyloidosis with trichiasis. Methods: Ophthalmological examination was performed in nine patients to determine secondary corneal amyloidosis with trichiasis. Congo red staining and immunohistochemistry using anti-human lactoferrin antibody were used for biopsied corneal samples. For genetic analyses, single strand conformation polymorphism (SSCP), direct DNA sequence analysis, and polymerase chain reaction (PCR) induced mutation restriction analysis (IMRA) were employed to detect lactoferrin gene polymorphism. Results: All patients had had trichiasis at least for 1 year, and all amyloid-like deposits were found in one eye with trichiasis. Ophthalmological examination revealed that eight patients showed gelatinous type of amyloid deposition and one showed lattice type of amyloid deposition. Studies of biopsied corneal samples with Congo red stain revealed positive staining just under the corneal epithelial cells. Immunoreactivity of anti-human lactoferrin antibodies was recognised in all tissues with positive Congo red staining. Lactoferrin gene analysis revealed that seven patients were heterozygotic and two were homozygotic for lactoferrin Glu561Asp. The frequency of the polymorphism in the patients was significantly different from that in 56 healthy control subjects. Conclusion: Lactoferrin Glu561Asp is a key polymorphism related to facilitating amyloid formation in corneal amyloidosis with trichiasis.

Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis

BACKGROUND Patisiran, an investigational RNA interference therapeutic agent, specifically inhibits hepatic synthesis of transthyretin. METHODS In this phase 3 trial, we randomly assigned patients with hereditary transthyretin amyloidosis with polyneuropathy, in a 2:1 ratio, to receive intravenous patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks. The primary end point was the change from baseline in the modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment) at 18 months. Other assessments included the Norfolk Quality of Life–Diabetic Neuropathy (Norfolk QOL‐DN) questionnaire (range, ‐4 to 136, with higher scores indicating worse quality of life), 10‐m walk test (with gait speed measured in meters per second), and modified body‐mass index (modified BMI, defined as [weight in kilograms divided by square of height in meters]×albumin level in grams per liter; lower values indicated worse nutritional status). RESULTS A total of 225 patients underwent randomization (148 to the patisiran group and 77 to the placebo group). The mean (±SD) mNIS+7 at baseline was 80.9±41.5 in the patisiran group and 74.6±37.0 in the placebo group; the least‐squares mean (±SE) change from baseline was ‐6.0±1.7 versus 28.0±2.6 (difference, ‐34.0 points; P<0.001) at 18 months. The mean (±SD) baseline Norfolk QOL‐DN score was 59.6±28.2 in the patisiran group and 55.5±24.3 in the placebo group; the least‐squares mean (±SE) change from baseline was ‐6.7±1.8 versus 14.4±2.7 (difference, ‐21.1 points; P<0.001) at 18 months. Patisiran also showed an effect on gait speed and modified BMI. At 18 months, the least‐squares mean change from baseline in gait speed was 0.08±0.02 m per second with patisiran versus ‐0.24±0.04 m per second with placebo (difference, 0.31 m per second; P<0.001), and the least‐squares mean change from baseline in the modified BMI was ‐3.7±9.6 versus ‐119.4±14.5 (difference, 115.7; P<0.001). Approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion‐related reactions; the overall incidence and types of adverse events were similar in the two groups. CONCLUSIONS In this trial, patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO ClinicalTrials.gov number, NCT01960348.)

Near-infrared spectrophotoscopy of finger venules in assessment of autonomic dysfunction

The authors evaluated morphologic changes in the venules of the finger using near-infrared spectrophotoscopy in patients with autonomic dysfunction, such as familial amyloidotic polyneuropathy and multiple-system atrophy. Abnormalities of the venules, such as tortuosity, irregular venous caliber, and microaneurysm-like change, and a linear negative correlation between the degree of orthostatic hypotension and the degree of vasoconstriction of the venules were observed.

Age-dependent increase in thiol conjugated forms of transthyretin (TTR) in the elderly: quantitative analyses by the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) protein chip system.

Senile systemic amyloidosis (SSA) caused by wild-type (WT) transthyretin (TTR) is a prevalent disorder in the elderly. However, mechanisms of WT TTR amyloid formation and risk factors for SSA remain to be elucidated. In this study, to determine age-related changes in serum TTR, we investigated TTR concentrations and modifications ratios in sera of the elderly. Neither significant age-related changes nor gender-related distinctions in serum TTR concentrations were observed from the age of 60’s to 90’s. In addition, serum TTR concentrations of SSA patients did not significantly change compared to those in coeval elderly. However, all kinds of thiol conjugated-TTR ratios significantly increased as the age by means of surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry, while those of SSA patients did not significantly differ from those of the coeval elderly. We speculate that those thiol conjugations of TTR are one of the required trigger for SSA in the elderly. Introduction: Senile systemic amyloidosis (SSA) is a sporadic transthyretin (TTR) amyloidosis derived from wild-type TTR [1]. It has been well documented that most of the patients with SSA show slowly progressive amyloid cardiomyopathy, which causes congestive heart failure and arrhythmia [1]. Several reports exhibited that the prevalence of SSA was up to 25% in the elderly older than 80 years of age based on examination of autopsy-derived cardiac specimens in the United States and Europe [2]. However, the pathogenesis of SSA remains to be elucidated. TTR is mainly produced in the liver and is present in plasma normally as a homotetramer. In analysis of serum samples, several conjugated forms of TTR were observed in addition to unconjugated forms of TTR [3]. Cys-10 residue in TTR is generally one of the modification sites, and the cys-conjugated form of TTR is found most frequently in serum [4,5]. In addition, other modifications of TTR, such as Ssulfonation, cysteinylglycylation, and glutathionylation, are also detected. It was reported in in vitro study that those modifications increased amyloidogenicity of TTR. However, age-dependent changes of such TTR modifications in the elderly and SSA patients were not still determined. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) combines chromatography with mass spectrometry and uses arrays with different surface chemistries. Chromatographic surfaces of ProteinChips provide good support for co-crystallization of matrix and target proteins, which results in formation of a homogeneous layer on the spot. SELDI-TOF MS instrument is also especially adapted for quantitative assay of TTR molecule [6]. In this study, to determine age-related changes in serum TTR, we investigated TTR concentrations and thiol conjugations of TTR in sera of the elderly by means of nephelometry and SELDI-TOF MS, respectively. Materials and methods: Serum samples and measurement of serum TTR concentrations: For measuring TTR concentrations, 409 serum samples of elderly patients (60’s: 136, 70’s: 140, 80’s: 114, and 90’s: 19) and 6 serum samples of SSA patients (74.5+ 5.5 years old) were employed. We excluded patients with low albumin concentrations (54.1 g/dl) and/or high C-reactive protein concentrations (40.3 mg/dl) in sera from the study. Serum TTR concentrations were measured by nephelometry. For evaluation of TTR modifications, we used each 10 serum samples of patients in 60’s, 70’s, 80’s and 90’s, respectively. Informed consent was obtained from each subject. All studies using serum samples were in accordance with the current revision of the Helsinki Declaration. Figure 1. Serum TTR concentrations in the elderly and SSA patients. Serum samples of 409 elderly patients and 6 SSA patients (74.5+ 5.5 years) were employed. Serum TTR concentrations were measured by nephelometry. 14

Effect of cyclodextrins on transthyretin amyloid formation in transthyretin-related amyloidosis.

Transthyretin (TTR), a beta-sheet rich protein, is a precursor protein of familial amyloidotic polyneuropathy (FAP). Although it has been widely accepted that protein misfolding of monomeric form of TTR is rate limiting for amyloid formation, no effective therapy targeting this misfolding step is available. In this study, we focused on cyclodextrins (CyDs), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various b-CyDs, GUGb-CyD showed potent inhibition of TTR amyloid formation. In the presence of GUG-b-CyD, no significant TTR amyloid fibrils were detected by electron microscopic analysis. Moreover, far-UV circular dichroism spectra analysis showed that GUG-b-CyD reduced the conformational change of TTR in the process of amyloid formation. Taken together, these data suggest that GUG-b-CyD may modify the stability of TTR conformation, which, in turn, leads to the suppression of TTR amyloid formation. Introduction: Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), which is induced by amyloidogenic TTR (ATTR), is characterized by systemic accumulation of amyloid fibrils [1]. It has been proposed that tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a non-native monomer with low conformational stability can lead to amyloid fibril formation [2]. Previous works have also shown that further structural change within the monomer caused by protein misfolding is a rate-limiting step to form TTR amyloid fibril aggregation [3]. However, no effective therapy targeting this misfolding step is available as of this moment. Cyclodextrins (CyDs), cyclic oligosaccharides composed of 6–8 glucose units, are widely used as prospective drug carriers in the pharmaceutical field [4]. There are three common types of natural CyDs depending on how many glucose units are present: aCyD (6), b-CyD (7), and g-CyD (8). Since CyD contains a central hydrophobic cavity and this cavity can serve as an inclusion site for hydrophobic molecules, CyDs are mainly used as multi-functional drug carriers by enhancing the bioavailability of lipophilic drugs, improving efficacy of drugs, and reducing side effects [5]. In addition, it has been proposed that b-CyDs improve the bioavailability of protein drug formulation. Recent studies revealed that b-CyDs bound to a hydrophobic part of the protein surface and increased its stability, which, in turn, led to prevent the protein misfolding and aggregation [6–8]. Because it is well documented that multiple hydrophobic regions of TTR are exposed in the process of TTR amyloid formation, these evidences suggest that b-CyDs may have potential to prevent TTR amyloid formation by inhibiting the misfolding of monomeric form of TTR. In this study, we elucidated the inhibitory effect of CyDs on TTR amyloid formation. Materials and methods: Both WT-TTR and ATTR-V30M were purified from serum samples obtained from healthy volunteers and homozygotic FAP ATTR V30M patients, respectively. b-CyD, hydroxpropyl-b-CyD (HP-b-CyD), 6-O-a-(4-O-a-DGlucuronyl)-D-glucosyl-b-CyD (GUG-b-CyD), and 6-O-a-maltosyl-b-CyD (G2-b-CyD) were examined in this study. To assess the effect of CyDs on TTR amyloid formation in vitro, thioflavin T-based fluorimetric assay was performed. The presence of TTR amyloid fibril was confirmed by electron microscopic analysis as described previously [9]. To evaluate the effect of CyDs on the conformational change of TTR, far-UV circular dichroism spectra analysis and fluorescent spectrum analysis were performed [10]. Results and discussion: Thioflavin T-based fluorimetric assay was first performed to assess the effect of possible b-CyDs on TTR amyloid formation. Of various b-CyDs, GUG-b-CyD showed potent inhibition of TTR amyloid formation, compared with b-CyD, HP-b-CyD, and G2-b-CyD. The amyloid formation of both WT-TTR and ATTR-V30M was suppressed by GUG-b-CyD in a dose-dependent manner. The effect of GUG-b-CyD was sustained and increased in a time-dependent manner, and the significant inhibition of TTR amyloid formation was observed even at 14 days after GUG-b-CyD administration. To confirm the inhibitory effect of GUG-bCyD further, electron microscopic analysis was performed. In agreement with the results described above, TTR amyloid formation was significantly suppressed in the presence of GUG-b-CyD. From these data, it is suggested that GUG-b-CyD indeed has potential to suppress TTR amyloid formation. To investigate the precise mechanism to how GUG-b-CyD inhibits TTR amyloid formation, the conformational change of TTR by GUG-b-CyD treatment was examined by far-UV CD spectra analysis. CD spectra of TTR were shifted and 58