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JNNP Online First, published on January 20, 2015 as 10.1136/jnnp-2014-308724 General neurology
REVIEW
Transthyretin (ATTR) amyloidosis: clinical spectrum, molecular pathogenesis and disease-modifying treatments Yoshiki Sekijima1,2 1
Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan 2 Institute for Biomedical Sciences, Shinshu University, Matsumoto, Japan Correspondence to Dr Yoshiki Sekijima, Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan; [email protected] Received 9 September 2014 Revised 22 December 2014 Accepted 25 December 2014
ABSTRACT Transthyretin (ATTR) amyloidosis is a life-threatening, gain-of-toxic-function disease characterised by extracellular deposition of amyloid fibrils composed of transthyretin (TTR). TTR protein destabilised by TTR gene mutation is prone to dissociate from its native tetramer to monomer, and to then misfold and aggregate into amyloid fibrils, resulting in autosomal dominant hereditary amyloidosis, including familial amyloid polyneuropathy, familial amyloid cardiomyopathy and familial leptomeningeal amyloidosis. Analogous misfolding of wild-type TTR results in senile systemic amyloidosis, now termed wild-type ATTR amyloidosis, characterised by acquired amyloid disease in the elderly. With the availability of genetic, biochemical and immunohistochemical diagnostic tests, patients with ATTR amyloidosis have been found in many nations; however, misdiagnosis is still common and considerable time is required before correct diagnosis in many cases. The current standard first-line treatment for hereditary ATTR amyloidosis is liver transplantation, which allows suppression of the main source of variant TTR. However, large numbers of patients are not suitable transplant candidates. Recently, the clinical effects of TTR tetramer stabilisers, diflunisal and tafamidis, were demonstrated in randomised clinical trials, and tafamidis has been approved for treatment of hereditary ATTR amyloidosis in European countries and in Japan. Moreover, antisense oligonucleotides and small interfering RNAs for suppression of variant and wild-type TTR synthesis are promising therapeutic approaches to ameliorate ATTR amyloidosis and are currently in phase III clinical trials. These newly developed therapies are expected to be effective for not only hereditary ATTR amyloidosis but also wild-type ATTR amyloidosis.
INTRODUCTION
To cite: Sekijima Y. J Neurol Neurosurg Psychiatry Published Online First: [please include Day Month Year] doi:10.1136/jnnp2014-308724
The amyloidoses are a group of gain-of-toxicfunction diseases caused by the aggregation of a specific protein. According to the official International Society of Amyloidosis (ISA) Amyloid Fibril Protein Nomenclature List,1 31 different amyloid fibril proteins have been identified in humans, and the diseases are now classified according to the nature of the amyloid precursor protein. Transthyretin (TTR) is a representative amyloidogenic protein in humans. Variant TTR deposition causes autosomal dominant hereditary ATTR amyloidosis. To date, more than 120 TTR gene mutations have been reported and considerable genotype–phenotype correlations have been identified.2 The three main
phenotypes of hereditary ATTR amyloidosis are familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) and familial leptomeningeal amyloidosis (table 1). On the other hand, wild-type ATTR deposition leads to an acquired amyloid disease, senile systemic amyloidosis (now termed wild-type ATTR (ATTRwt) amyloidosis), which typically presents later than hereditary ATTR amyloidosis.3 4 ATTR amyloidosis was historically considered to be a rare endemic disease. However, recent progress in diagnosis indicates that there are many patients with hereditary ATTR amyloidosis worldwide. Furthermore, ATTRwt amyloidosis is thought to be a common ageing-related disorder, as more than 10% of people over the age of 80 have wild-type TTR deposition in postmortem studies.3 5 ATTR amyloidosis used to be an incurable disease, but liver transplantation has been shown to be effective for FAP.6 While liver transplantation markedly improves the prognosis of patients with FAP, large numbers of patients are not suitable transplant candidates because of their age and/or advanced disease status. In addition, transplantation is not a viable option for FAC, familial leptomeningeal amyloidosis and ATTRwt amyloidosis. Therefore, it is desirable to develop more general alternative therapeutic strategies to ameliorate ATTR amyloidosis. Recently, the molecular, biological and chemical pathogeneses of ATTR amyloidosis have been clarified,7 8 and several novel disease-modifying treatments have been developed.9 10 Among these, the TTR tetramer stabilisers, diflunisal11 and tafamidis,12 have been shown to inhibit progression of polyneuropathy and preserve quality of life in patients with FAP in randomised controlled trials. This review focuses on recent progress in our understanding of the clinical spectrum, molecular pathogenesis and development of novel therapeutic approaches for ATTR amyloidosis.
CLINICAL SPECTRUM OF ATTR AMYLOIDOSIS Hereditary ATTR amyloidosis Hereditary ATTR amyloidosis is the most common form of hereditary amyloidosis caused by TTR gene mutation. Hereditary ATTR amyloidosis is a lifethreatening, gain-of-toxic-function disease that may present with peripheral neuropathy, autonomic neuropathy, cardiomyopathy, ophthalmopathy and/ or leptomeningeal amyloidosis. FAP, FAC and familial leptomeningeal amyloidosis are the three main phenotypes of hereditary ATTR amyloidosis (table 1). However, the phenotype is not always uniform, even in patients with the same mutation.
Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
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General neurology Table 1 Clinical spectrum of transthyretin (ATTR) amyloidosis Precursor proteins
Clinical Phenotypes
Representative TTR genotypes
Effective diseasemodifying therapies
Ongoing clinical trials
Variant TTR
Hereditary ATTR amyloidosis Familial amyloid polyneuropathy (FAP)
Val30Met (p.Val50Met) Leu58His (p.Leu78His)
Liver transplantation Tafamidis Diflunisal
ASOs* (ISIS-TTRRX, Phase III) siRNA** (ALN-TTR02, Phase III) Doxy-TUDCA*** (Phase II)
Familial amyloid cardiomyopathy (FAC)
Val122Ile (p.Val142Ile) Leu111Met (Leu131Met)
Combined heart and liver transplantation
Tafamidis (Phase III) siRNA** (ALN-TTRsc, Phase II)
Familial leptomeningeal amyloidosis
Asp18Gly (p.Asp38Gly) Ala25Thr (p. Ala45Thr)
None
None
Wild-type
None
Tafamidis (Phase III) siRNA** (ALN-TTRsc, Phase II)
Wild-type TTR
Wild-type ATTR (ATTRwt) amyloidosis
* ASOs, Antisense oligonucleotides; **siRNA, Small interfering RNA; *** Doxy-TUDCA, combined use of doxycycline and tauroursodeoxycholic acid.
For example, most patients with Val30Met ( p.Val50Met) mutation develop both neuropathy and cardiomyopathy during the course of the disease. Cardiomyopathy could be an initial and/ or main symptom in patients with Val30Met mutation, although FAP is the most common phenotype in such cases.
Familial amyloid polyneuropathy FAP is the most common clinical phenotype of hereditary ATTR amyloidosis. The Val30Met mutation, found worldwide, is the most common TTR mutation and is responsible for the wellknown large foci of patients with FAP in Portugal, Sweden and Japan.2 Patients with Val30Met mutation (ATTR Val30Met) are classified into two groups, early-onset and late-onset. Early-onset ATTR Val30Met patients are seen in the endemic foci in Portugal and Japan, and are characterised by onset before the age of 50 years, high penetrance rate, predominant loss of superficial sensation including nociception and thermal sensation, and marked autonomic dysfunction, including orthostatic hypotension, sexual impotence, neurogenic bladder and disturbed bowel movement. In addition, various types of cardiac conduction blocks frequently appear, requiring the implantation of a pacemaker.2 In contrast to the early-onset patients, ATTR Val30Met patients in non-endemic areas and the endemic focus in Sweden typically have onset at a late age, usually after the age of 60 (late-onset ATTR Val30Met). The clinical characteristics of late-onset ATTR Val30Met include a low penetrance rate, often with lack of a family history, loss of all sensory modalities, relatively mild autonomic dysfunction and male predominance.13 Amyloid cardiomyopathy is also common in late-onset ATTR Val30Met.13 The basis of this variability is uncertain and may be attributable to other genetic factors, epigenetic factors, epistasis or environmental factors. In patients with specific TTR mutations, such as Leu58His (p.Leu78His), Ile84Ser (p.Ile104Ser) and Tyr114His (p.Tyr134His), neuropathy starts in the upper extremities as carpal tunnel syndrome.2
Other representative TTR mutations responsible for FAC include Ser50Ile ( p.Ser70Ile), Thr60Ala ( p.Thr80Ala), Ile68Leu (p.Ile88Leu) and Leu111Met ( p.Leu131Met).2 Patients with FAC show congestive heart failure, intractable arrhythmia and conduction blocks and occasionally require implantation of a pacemaker and/or implantable cardioverter defibrillator. Typical ECG findings of patients with FAC include low voltage in the standard limb leads and QS pattern in the right precordial leads with conduction blocks. Symmetrical thickening of interventricular septum and ventricular walls is seen on ultrasound and MRI. Global or patchy subendocardial late gadolinium enhancement of the myocardium is also a characteristic MRI finding in cases of cardiac amyloidosis. Left ventricular systolic function is usually preserved in the early stage of the disease. Technetium-99 m pyrophosphate (99mTc-PYP), 99m Tc-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) and 99mTc-hydroxy methylene diphosphonate (99mTc-HPD) myocardial scintigraphy are also valuable for detecting cardiac ATTR amyloid deposition.
Familial leptomeningeal amyloidosis Cerebral amyloid angiopathy accompanied by leptomeningeal amyloidosis are the main clinical features of patients with hereditary ATTR amyloidosis with several specific TTR gene mutations, such as Asp18Gly ( p.Asp38Gly), Ala25Thr ( p.Ala45Thr) and Tyr114Cys ( p.Tyr134Cys).2 In this disease, the main source of variant TTR is the choroid plexus and amyloid deposition is observed in the media and adventitia of medium-sized and small arteries, arterioles and veins of the cortex and leptomeninges. These pathological changes induce cerebral infarction, cerebral haemorrhage, subarachnoid haemorrhage and/or hydrocephalus, and cause various central nervous dysfunctions, such as spastic paralysis, ataxia, convulsions and dementia. Leptomeningeal amyloidosis develops only rarely in ATTR Val30Met patients, but can be a serious complication in patients with posttransplant, as choroid plexus continues to produce variant TTR after transplantation.16
Familial amyloid cardiomyopathy FAC is another common clinical phenotype of hereditary ATTR amyloidosis, and Val122Ile ( p.Val142Ile) is the most common mutation responsible for this disease. It was reported that ≥3% of African-Americans are heterozygous for Val122Ile mutation and develop late-onset cardiac amyloidosis,14 15 although the exact penetrance is unknown. The frequencies of Val122Ile in Caucasian, Hispanic and Asian populations are very low.15 2
Other organ involvement in hereditary ATTR amyloidosis Ocular involvement, including vitreous opacity, glaucoma, dry eye and ocular amyloid angiopathy, is common and occurs in most hereditary patients with ATTR amyloidosis during the course of the disease.17 Gastrointestinal symptoms, including recurrent vomiting, constipation and/or severe watery diarrhoea are frequently observed Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
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General neurology in hereditary patients with ATTR amyloidosis, especially in early-onset ATTR Val30Met patients in endemic foci.18 Renal involvement, including nephritic syndrome and progressive renal failure, occurs in about one-third of patients in Portugal;19 however, severe renal dysfunction rarely occurs in patients with late-onset disease.
Wild-type ATTR (ATTRwt) amyloidosis Wild-type ATTR deposition in systemic organs is thought to be a common ageing-related phenomenon similar to Aβ deposition in the brain, as post-mortem studies showed that more than 10% of people over the age of 80 have ATTR amyloid deposition.3 5 However, only a limited number of ATTRwt amyloidosis patients have been reported to date, and the exact prevalence of the disease is unknown, mainly because (1) a substantial amount of wild-type TTR deposition is thought to be necessary to develop clinical symptoms or signs, and (2) ATTRwt amyloidosis is markedly underdiagnosed. Typically, patients with ATTRwt amyloidosis show cardiac manifestations, such as congestive heart failure, atrial fibrillation and intractable arrhythmia, and occasionally require implantation of an implantable cardioverter defibrillator. Carpal tunnel syndrome is another common clinical manifestation of ATTRwt amyloidosis and develops as an initial symptom of the disease in many patients.20 Cardiogenic embolism and mild to moderate renal dysfunction are also frequently seen in such cases. There are no effective disease-modifying therapies for ATTRwt amyloidosis, and mean survival period from the onset of congestive heart failure symptoms is 75 months.
DIAGNOSIS OF ATTR AMYLOIDOSIS Diagnosis of hereditary ATTR amyloidosis In addition to the clinical symptoms described above, proven amyloid deposition in biopsy specimens and identification of disease-causing mutations in the TTR gene are necessary to establish the diagnosis. Deposition of amyloid in tissue can be demonstrated by Congo red staining of biopsy materials. Tissues suitable for biopsy include the subcutaneous fatty tissue of the abdominal wall and gastroduodenal mucosa. Amyloid deposition can also be detected in the sural nerve, rectal mucosa, salivary glands and endomyocardial biopsy specimens, as well as tenosynovial tissues obtained at carpal tunnel release surgery. Ideally, immunocytochemical study of the amyloid-positive tissue should be performed to confirm the presence of amyloid precursor protein. It should be noted that negative biopsy findings do not rule out amyloidosis because amyloid deposition is often patchy.2 21 22 Therefore, TTR gene analysis should be performed in parallel with tissue biopsy when hereditary ATTR amyloidosis is suspected. Hereditary ATTR amyloidosis can be excluded if genetic testing for TTR yields a negative result (no mutation).
Diagnosis of wild-type ATTR (ATTRwt) amyloidosis Clinical manifestations, proven ATTR amyloid deposition in biopsy specimens and confirmation of wild-type TTR genotype are necessary to establish the diagnosis of ATTRwt amyloidosis. Immunocytochemical or proteomic analysis of the amyloid positive tissue is essential for the diagnosis of ATTRwt amyloidosis to confirm the type of amyloid protein involved in the disease. In most diagnosed cases, tissue samples are obtained by endomyocardial biopsy; however, this is rarely performed due to its high degree of invasiveness. Abnormal uptake of 99mTc in myocardial scintigraphy is highly suggestive of ATTR cardiac amyloidosis (see FAC), although histopathological confirmation is Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
necessary. Surgical skin biopsy including the deep subcutaneous fat pad is a useful alternative histopathological tool for diagnosis of ATTRwt amyloidosis.23 If wild-type TTR-derived amyloid deposition is found in tenosynovial tissues obtained at carpal tunnel surgery, additional screening for amyloid deposition is necessary to confirm ‘systemic ATTRwt amyloidosis’, as wild-type TTR derived amyloid deposition localised to tenosynovial tissues, ‘localised ATTRwt amyloidosis’, is common in elderly men.20
MOLECULAR PATHOGENESIS OF ATTR AMYLOIDOSIS Structure and function of TTR TTR is a 55 kDa homotetrameric protein composed of 127-residue β-sheet-rich subunits synthesised in and secreted by the liver into the bloodstream.24 The main physiological functions of TTR are transport of thyroxine (T4) and retinol binding protein–vitamin A complex (holoRBP). The TTR tetramer contains two identical T4-binding sites located in a channel at the centre of the molecule.24 However, a very small proportion (80%.42 Survival analyses also demonstrated that TTR genotype is one of the most influential factors, as the 5-year and 10-year patient survival rates in the Val30Met group (82% and 74%, respectively) were significantly better than those in the non-Val30Met group (59% and 44%, respectively).42 43 Although prognosis of transplanted early-onset ATTR Val30Met patients is excellent,44–46 survival of transplanted patients does not differ from that of non-transplanted patients in late-onset ATTR Val30Met.43 45 It was also reported that female transplanted late-onset ATTR Val30Met patients had significantly improved survival compared with corresponding male patients.45 Progression of cardiac amyloid deposition is of more significance in non-Val30Met and late-onset Val30Met transplant recipients, which largely explains the less favourable long-term survival rates in these groups.45 The effects of liver transplantation on neuropathy were evident as progression of autonomic and peripheral neuropathy was stopped or even slightly improved in most patients.46 While liver transplantation is the current standard therapeutic strategy for FAP, it has several limitations, including the requirement for surgery, long-term post-transplantation immunosuppressive therapy and progression of eye, cardiac and leptomeningeal amyloidosis after transplantation. Eye and leptomeningeal deposition of TTR are not relieved by liver transplantation due to TTR synthesis by the retinal pigment epithelium and choroid plexus. On the other hand, cardiac amyloidosis progresses in some patients even after liver transplantation, because wild-type TTR deposition often continues.47 Furthermore, large numbers of patients are not suitable transplant candidates because of their age and/or advanced disease status. Finally, transplantation is not a viable option for FAC, familial leptomeningeal amyloidosis and ATTRwt amyloidosis.3 4 Therefore, it is necessary to develop more general, convenient and non-invasive alternative therapies for ATTR amyloidosis.
Figure 1 Mechanism of ATTR amyloid fibril formation and sites of action of therapeutic strategies. *The clinical effectiveness of antisense oligonucleotides, small interfering RNA, combined use of doxycycline and tauroursodeoxycholic acid, and immune therapy has not yet been proven. TTR, transthyretin. 4
Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
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General neurology TTR tetramer stabiliser Another therapeutic option for ameliorating ATTR amyloidosis is to stabilise the native TTR tetramer structure, as tetramer dissociation is necessary for ATTR amyloid fibril formation (figure 1). In fact, a trans-suppressor mutation, Thr119Met ( p.Thr139Met), was shown to prevent disease in ATTR Val30Met/Thr119Met compound heterozygotes by stabilising the tetrameric native state.48 On the other hand, it was reported that binding of T4, a natural TTR ligand, to two sites in the TTR tetramer, stabilises the native state over the dissociative transition state and thus inhibits amyloidogenesis in vitro. However, more than 99% of the T4 binding sites are unoccupied in human serum, as described above (see MOLECULAR PATHOGENESIS OF ATTR AMYLOIDOSIS). Based on these findings, Kelly and co-workers performed robust screening and structure-based drug design to find small molecules that bind tightly and selectively to the TTR tetramer.49 50 Typical TTR stabilisers have two aromatic substructures and a linker, and two small molecules, diflunisal and tafamidis, have been shown to slow the rate of disease progression of FAP in randomised clinical trials10–12 (table 1, figure 1).
Diflunisal Diflunisal, a salicylic acid derivative, is a well-known oral nonsteroidal anti-inflammatory drug that was developed more than 40 years ago. Previous in vitro analysis49 50 showed that diflunisal is able to bind to the T4 binding sites of TTR (Kd1=75 nM, Kd2=1.1 μM) and inhibit ATTR amyloid fibril formation at relatively low concentrations. Based on these findings, phase I51 and II52 clinical trials of diflunisal were conducted and showed that diflunisal at a dose of 250 mg twice daily successfully complexed to the T4 binding sites and stabilised circulating TTR tetramers. Subsequently, Berk and co-workers conducted an investigator-initiated randomised double-blind placebocontrolled multicentre phase III trial of diflunisal from 2006 through 2012.11 In this study, 130 patients with FAP (mean age 59.7 years; Val30Met 54.6%, non-Val30Met 45.4%) were enrolled and randomly assigned to receive diflunisal at 250 mg or placebo twice daily for 2 years. This phase III trial demonstrated the clinical effect of diflunisal on polyneuropathy progression, as the primary endpoint, change from baseline in the Neuropathy Impairment Score plus 7 nerve test (NIS+7), deteriorated by 8.7 points in the diflunisal group and 25.0 points in the placebo group ( p
JNNP Online First, published on January 20, 2015 as 10.1136/jnnp-2014-308724 General neurology
REVIEW
Transthyretin (ATTR) amyloidosis: clinical spectrum, molecular pathogenesis and disease-modifying treatments Yoshiki Sekijima1,2 1
Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan 2 Institute for Biomedical Sciences, Shinshu University, Matsumoto, Japan Correspondence to Dr Yoshiki Sekijima, Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan; [email protected] Received 9 September 2014 Revised 22 December 2014 Accepted 25 December 2014
ABSTRACT Transthyretin (ATTR) amyloidosis is a life-threatening, gain-of-toxic-function disease characterised by extracellular deposition of amyloid fibrils composed of transthyretin (TTR). TTR protein destabilised by TTR gene mutation is prone to dissociate from its native tetramer to monomer, and to then misfold and aggregate into amyloid fibrils, resulting in autosomal dominant hereditary amyloidosis, including familial amyloid polyneuropathy, familial amyloid cardiomyopathy and familial leptomeningeal amyloidosis. Analogous misfolding of wild-type TTR results in senile systemic amyloidosis, now termed wild-type ATTR amyloidosis, characterised by acquired amyloid disease in the elderly. With the availability of genetic, biochemical and immunohistochemical diagnostic tests, patients with ATTR amyloidosis have been found in many nations; however, misdiagnosis is still common and considerable time is required before correct diagnosis in many cases. The current standard first-line treatment for hereditary ATTR amyloidosis is liver transplantation, which allows suppression of the main source of variant TTR. However, large numbers of patients are not suitable transplant candidates. Recently, the clinical effects of TTR tetramer stabilisers, diflunisal and tafamidis, were demonstrated in randomised clinical trials, and tafamidis has been approved for treatment of hereditary ATTR amyloidosis in European countries and in Japan. Moreover, antisense oligonucleotides and small interfering RNAs for suppression of variant and wild-type TTR synthesis are promising therapeutic approaches to ameliorate ATTR amyloidosis and are currently in phase III clinical trials. These newly developed therapies are expected to be effective for not only hereditary ATTR amyloidosis but also wild-type ATTR amyloidosis.
INTRODUCTION
To cite: Sekijima Y. J Neurol Neurosurg Psychiatry Published Online First: [please include Day Month Year] doi:10.1136/jnnp2014-308724
The amyloidoses are a group of gain-of-toxicfunction diseases caused by the aggregation of a specific protein. According to the official International Society of Amyloidosis (ISA) Amyloid Fibril Protein Nomenclature List,1 31 different amyloid fibril proteins have been identified in humans, and the diseases are now classified according to the nature of the amyloid precursor protein. Transthyretin (TTR) is a representative amyloidogenic protein in humans. Variant TTR deposition causes autosomal dominant hereditary ATTR amyloidosis. To date, more than 120 TTR gene mutations have been reported and considerable genotype–phenotype correlations have been identified.2 The three main
phenotypes of hereditary ATTR amyloidosis are familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC) and familial leptomeningeal amyloidosis (table 1). On the other hand, wild-type ATTR deposition leads to an acquired amyloid disease, senile systemic amyloidosis (now termed wild-type ATTR (ATTRwt) amyloidosis), which typically presents later than hereditary ATTR amyloidosis.3 4 ATTR amyloidosis was historically considered to be a rare endemic disease. However, recent progress in diagnosis indicates that there are many patients with hereditary ATTR amyloidosis worldwide. Furthermore, ATTRwt amyloidosis is thought to be a common ageing-related disorder, as more than 10% of people over the age of 80 have wild-type TTR deposition in postmortem studies.3 5 ATTR amyloidosis used to be an incurable disease, but liver transplantation has been shown to be effective for FAP.6 While liver transplantation markedly improves the prognosis of patients with FAP, large numbers of patients are not suitable transplant candidates because of their age and/or advanced disease status. In addition, transplantation is not a viable option for FAC, familial leptomeningeal amyloidosis and ATTRwt amyloidosis. Therefore, it is desirable to develop more general alternative therapeutic strategies to ameliorate ATTR amyloidosis. Recently, the molecular, biological and chemical pathogeneses of ATTR amyloidosis have been clarified,7 8 and several novel disease-modifying treatments have been developed.9 10 Among these, the TTR tetramer stabilisers, diflunisal11 and tafamidis,12 have been shown to inhibit progression of polyneuropathy and preserve quality of life in patients with FAP in randomised controlled trials. This review focuses on recent progress in our understanding of the clinical spectrum, molecular pathogenesis and development of novel therapeutic approaches for ATTR amyloidosis.
CLINICAL SPECTRUM OF ATTR AMYLOIDOSIS Hereditary ATTR amyloidosis Hereditary ATTR amyloidosis is the most common form of hereditary amyloidosis caused by TTR gene mutation. Hereditary ATTR amyloidosis is a lifethreatening, gain-of-toxic-function disease that may present with peripheral neuropathy, autonomic neuropathy, cardiomyopathy, ophthalmopathy and/ or leptomeningeal amyloidosis. FAP, FAC and familial leptomeningeal amyloidosis are the three main phenotypes of hereditary ATTR amyloidosis (table 1). However, the phenotype is not always uniform, even in patients with the same mutation.
Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Downloaded from http://jnnp.bmj.com/ on April 11, 2015 - Published by group.bmj.com
General neurology Table 1 Clinical spectrum of transthyretin (ATTR) amyloidosis Precursor proteins
Clinical Phenotypes
Representative TTR genotypes
Effective diseasemodifying therapies
Ongoing clinical trials
Variant TTR
Hereditary ATTR amyloidosis Familial amyloid polyneuropathy (FAP)
Val30Met (p.Val50Met) Leu58His (p.Leu78His)
Liver transplantation Tafamidis Diflunisal
ASOs* (ISIS-TTRRX, Phase III) siRNA** (ALN-TTR02, Phase III) Doxy-TUDCA*** (Phase II)
Familial amyloid cardiomyopathy (FAC)
Val122Ile (p.Val142Ile) Leu111Met (Leu131Met)
Combined heart and liver transplantation
Tafamidis (Phase III) siRNA** (ALN-TTRsc, Phase II)
Familial leptomeningeal amyloidosis
Asp18Gly (p.Asp38Gly) Ala25Thr (p. Ala45Thr)
None
None
Wild-type
None
Tafamidis (Phase III) siRNA** (ALN-TTRsc, Phase II)
Wild-type TTR
Wild-type ATTR (ATTRwt) amyloidosis
* ASOs, Antisense oligonucleotides; **siRNA, Small interfering RNA; *** Doxy-TUDCA, combined use of doxycycline and tauroursodeoxycholic acid.
For example, most patients with Val30Met ( p.Val50Met) mutation develop both neuropathy and cardiomyopathy during the course of the disease. Cardiomyopathy could be an initial and/ or main symptom in patients with Val30Met mutation, although FAP is the most common phenotype in such cases.
Familial amyloid polyneuropathy FAP is the most common clinical phenotype of hereditary ATTR amyloidosis. The Val30Met mutation, found worldwide, is the most common TTR mutation and is responsible for the wellknown large foci of patients with FAP in Portugal, Sweden and Japan.2 Patients with Val30Met mutation (ATTR Val30Met) are classified into two groups, early-onset and late-onset. Early-onset ATTR Val30Met patients are seen in the endemic foci in Portugal and Japan, and are characterised by onset before the age of 50 years, high penetrance rate, predominant loss of superficial sensation including nociception and thermal sensation, and marked autonomic dysfunction, including orthostatic hypotension, sexual impotence, neurogenic bladder and disturbed bowel movement. In addition, various types of cardiac conduction blocks frequently appear, requiring the implantation of a pacemaker.2 In contrast to the early-onset patients, ATTR Val30Met patients in non-endemic areas and the endemic focus in Sweden typically have onset at a late age, usually after the age of 60 (late-onset ATTR Val30Met). The clinical characteristics of late-onset ATTR Val30Met include a low penetrance rate, often with lack of a family history, loss of all sensory modalities, relatively mild autonomic dysfunction and male predominance.13 Amyloid cardiomyopathy is also common in late-onset ATTR Val30Met.13 The basis of this variability is uncertain and may be attributable to other genetic factors, epigenetic factors, epistasis or environmental factors. In patients with specific TTR mutations, such as Leu58His (p.Leu78His), Ile84Ser (p.Ile104Ser) and Tyr114His (p.Tyr134His), neuropathy starts in the upper extremities as carpal tunnel syndrome.2
Other representative TTR mutations responsible for FAC include Ser50Ile ( p.Ser70Ile), Thr60Ala ( p.Thr80Ala), Ile68Leu (p.Ile88Leu) and Leu111Met ( p.Leu131Met).2 Patients with FAC show congestive heart failure, intractable arrhythmia and conduction blocks and occasionally require implantation of a pacemaker and/or implantable cardioverter defibrillator. Typical ECG findings of patients with FAC include low voltage in the standard limb leads and QS pattern in the right precordial leads with conduction blocks. Symmetrical thickening of interventricular septum and ventricular walls is seen on ultrasound and MRI. Global or patchy subendocardial late gadolinium enhancement of the myocardium is also a characteristic MRI finding in cases of cardiac amyloidosis. Left ventricular systolic function is usually preserved in the early stage of the disease. Technetium-99 m pyrophosphate (99mTc-PYP), 99m Tc-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) and 99mTc-hydroxy methylene diphosphonate (99mTc-HPD) myocardial scintigraphy are also valuable for detecting cardiac ATTR amyloid deposition.
Familial leptomeningeal amyloidosis Cerebral amyloid angiopathy accompanied by leptomeningeal amyloidosis are the main clinical features of patients with hereditary ATTR amyloidosis with several specific TTR gene mutations, such as Asp18Gly ( p.Asp38Gly), Ala25Thr ( p.Ala45Thr) and Tyr114Cys ( p.Tyr134Cys).2 In this disease, the main source of variant TTR is the choroid plexus and amyloid deposition is observed in the media and adventitia of medium-sized and small arteries, arterioles and veins of the cortex and leptomeninges. These pathological changes induce cerebral infarction, cerebral haemorrhage, subarachnoid haemorrhage and/or hydrocephalus, and cause various central nervous dysfunctions, such as spastic paralysis, ataxia, convulsions and dementia. Leptomeningeal amyloidosis develops only rarely in ATTR Val30Met patients, but can be a serious complication in patients with posttransplant, as choroid plexus continues to produce variant TTR after transplantation.16
Familial amyloid cardiomyopathy FAC is another common clinical phenotype of hereditary ATTR amyloidosis, and Val122Ile ( p.Val142Ile) is the most common mutation responsible for this disease. It was reported that ≥3% of African-Americans are heterozygous for Val122Ile mutation and develop late-onset cardiac amyloidosis,14 15 although the exact penetrance is unknown. The frequencies of Val122Ile in Caucasian, Hispanic and Asian populations are very low.15 2
Other organ involvement in hereditary ATTR amyloidosis Ocular involvement, including vitreous opacity, glaucoma, dry eye and ocular amyloid angiopathy, is common and occurs in most hereditary patients with ATTR amyloidosis during the course of the disease.17 Gastrointestinal symptoms, including recurrent vomiting, constipation and/or severe watery diarrhoea are frequently observed Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
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General neurology in hereditary patients with ATTR amyloidosis, especially in early-onset ATTR Val30Met patients in endemic foci.18 Renal involvement, including nephritic syndrome and progressive renal failure, occurs in about one-third of patients in Portugal;19 however, severe renal dysfunction rarely occurs in patients with late-onset disease.
Wild-type ATTR (ATTRwt) amyloidosis Wild-type ATTR deposition in systemic organs is thought to be a common ageing-related phenomenon similar to Aβ deposition in the brain, as post-mortem studies showed that more than 10% of people over the age of 80 have ATTR amyloid deposition.3 5 However, only a limited number of ATTRwt amyloidosis patients have been reported to date, and the exact prevalence of the disease is unknown, mainly because (1) a substantial amount of wild-type TTR deposition is thought to be necessary to develop clinical symptoms or signs, and (2) ATTRwt amyloidosis is markedly underdiagnosed. Typically, patients with ATTRwt amyloidosis show cardiac manifestations, such as congestive heart failure, atrial fibrillation and intractable arrhythmia, and occasionally require implantation of an implantable cardioverter defibrillator. Carpal tunnel syndrome is another common clinical manifestation of ATTRwt amyloidosis and develops as an initial symptom of the disease in many patients.20 Cardiogenic embolism and mild to moderate renal dysfunction are also frequently seen in such cases. There are no effective disease-modifying therapies for ATTRwt amyloidosis, and mean survival period from the onset of congestive heart failure symptoms is 75 months.
DIAGNOSIS OF ATTR AMYLOIDOSIS Diagnosis of hereditary ATTR amyloidosis In addition to the clinical symptoms described above, proven amyloid deposition in biopsy specimens and identification of disease-causing mutations in the TTR gene are necessary to establish the diagnosis. Deposition of amyloid in tissue can be demonstrated by Congo red staining of biopsy materials. Tissues suitable for biopsy include the subcutaneous fatty tissue of the abdominal wall and gastroduodenal mucosa. Amyloid deposition can also be detected in the sural nerve, rectal mucosa, salivary glands and endomyocardial biopsy specimens, as well as tenosynovial tissues obtained at carpal tunnel release surgery. Ideally, immunocytochemical study of the amyloid-positive tissue should be performed to confirm the presence of amyloid precursor protein. It should be noted that negative biopsy findings do not rule out amyloidosis because amyloid deposition is often patchy.2 21 22 Therefore, TTR gene analysis should be performed in parallel with tissue biopsy when hereditary ATTR amyloidosis is suspected. Hereditary ATTR amyloidosis can be excluded if genetic testing for TTR yields a negative result (no mutation).
Diagnosis of wild-type ATTR (ATTRwt) amyloidosis Clinical manifestations, proven ATTR amyloid deposition in biopsy specimens and confirmation of wild-type TTR genotype are necessary to establish the diagnosis of ATTRwt amyloidosis. Immunocytochemical or proteomic analysis of the amyloid positive tissue is essential for the diagnosis of ATTRwt amyloidosis to confirm the type of amyloid protein involved in the disease. In most diagnosed cases, tissue samples are obtained by endomyocardial biopsy; however, this is rarely performed due to its high degree of invasiveness. Abnormal uptake of 99mTc in myocardial scintigraphy is highly suggestive of ATTR cardiac amyloidosis (see FAC), although histopathological confirmation is Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
necessary. Surgical skin biopsy including the deep subcutaneous fat pad is a useful alternative histopathological tool for diagnosis of ATTRwt amyloidosis.23 If wild-type TTR-derived amyloid deposition is found in tenosynovial tissues obtained at carpal tunnel surgery, additional screening for amyloid deposition is necessary to confirm ‘systemic ATTRwt amyloidosis’, as wild-type TTR derived amyloid deposition localised to tenosynovial tissues, ‘localised ATTRwt amyloidosis’, is common in elderly men.20
MOLECULAR PATHOGENESIS OF ATTR AMYLOIDOSIS Structure and function of TTR TTR is a 55 kDa homotetrameric protein composed of 127-residue β-sheet-rich subunits synthesised in and secreted by the liver into the bloodstream.24 The main physiological functions of TTR are transport of thyroxine (T4) and retinol binding protein–vitamin A complex (holoRBP). The TTR tetramer contains two identical T4-binding sites located in a channel at the centre of the molecule.24 However, a very small proportion (80%.42 Survival analyses also demonstrated that TTR genotype is one of the most influential factors, as the 5-year and 10-year patient survival rates in the Val30Met group (82% and 74%, respectively) were significantly better than those in the non-Val30Met group (59% and 44%, respectively).42 43 Although prognosis of transplanted early-onset ATTR Val30Met patients is excellent,44–46 survival of transplanted patients does not differ from that of non-transplanted patients in late-onset ATTR Val30Met.43 45 It was also reported that female transplanted late-onset ATTR Val30Met patients had significantly improved survival compared with corresponding male patients.45 Progression of cardiac amyloid deposition is of more significance in non-Val30Met and late-onset Val30Met transplant recipients, which largely explains the less favourable long-term survival rates in these groups.45 The effects of liver transplantation on neuropathy were evident as progression of autonomic and peripheral neuropathy was stopped or even slightly improved in most patients.46 While liver transplantation is the current standard therapeutic strategy for FAP, it has several limitations, including the requirement for surgery, long-term post-transplantation immunosuppressive therapy and progression of eye, cardiac and leptomeningeal amyloidosis after transplantation. Eye and leptomeningeal deposition of TTR are not relieved by liver transplantation due to TTR synthesis by the retinal pigment epithelium and choroid plexus. On the other hand, cardiac amyloidosis progresses in some patients even after liver transplantation, because wild-type TTR deposition often continues.47 Furthermore, large numbers of patients are not suitable transplant candidates because of their age and/or advanced disease status. Finally, transplantation is not a viable option for FAC, familial leptomeningeal amyloidosis and ATTRwt amyloidosis.3 4 Therefore, it is necessary to develop more general, convenient and non-invasive alternative therapies for ATTR amyloidosis.
Figure 1 Mechanism of ATTR amyloid fibril formation and sites of action of therapeutic strategies. *The clinical effectiveness of antisense oligonucleotides, small interfering RNA, combined use of doxycycline and tauroursodeoxycholic acid, and immune therapy has not yet been proven. TTR, transthyretin. 4
Sekijima Y. J Neurol Neurosurg Psychiatry 2015;0:1–8. doi:10.1136/jnnp-2014-308724
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General neurology TTR tetramer stabiliser Another therapeutic option for ameliorating ATTR amyloidosis is to stabilise the native TTR tetramer structure, as tetramer dissociation is necessary for ATTR amyloid fibril formation (figure 1). In fact, a trans-suppressor mutation, Thr119Met ( p.Thr139Met), was shown to prevent disease in ATTR Val30Met/Thr119Met compound heterozygotes by stabilising the tetrameric native state.48 On the other hand, it was reported that binding of T4, a natural TTR ligand, to two sites in the TTR tetramer, stabilises the native state over the dissociative transition state and thus inhibits amyloidogenesis in vitro. However, more than 99% of the T4 binding sites are unoccupied in human serum, as described above (see MOLECULAR PATHOGENESIS OF ATTR AMYLOIDOSIS). Based on these findings, Kelly and co-workers performed robust screening and structure-based drug design to find small molecules that bind tightly and selectively to the TTR tetramer.49 50 Typical TTR stabilisers have two aromatic substructures and a linker, and two small molecules, diflunisal and tafamidis, have been shown to slow the rate of disease progression of FAP in randomised clinical trials10–12 (table 1, figure 1).
Diflunisal Diflunisal, a salicylic acid derivative, is a well-known oral nonsteroidal anti-inflammatory drug that was developed more than 40 years ago. Previous in vitro analysis49 50 showed that diflunisal is able to bind to the T4 binding sites of TTR (Kd1=75 nM, Kd2=1.1 μM) and inhibit ATTR amyloid fibril formation at relatively low concentrations. Based on these findings, phase I51 and II52 clinical trials of diflunisal were conducted and showed that diflunisal at a dose of 250 mg twice daily successfully complexed to the T4 binding sites and stabilised circulating TTR tetramers. Subsequently, Berk and co-workers conducted an investigator-initiated randomised double-blind placebocontrolled multicentre phase III trial of diflunisal from 2006 through 2012.11 In this study, 130 patients with FAP (mean age 59.7 years; Val30Met 54.6%, non-Val30Met 45.4%) were enrolled and randomly assigned to receive diflunisal at 250 mg or placebo twice daily for 2 years. This phase III trial demonstrated the clinical effect of diflunisal on polyneuropathy progression, as the primary endpoint, change from baseline in the Neuropathy Impairment Score plus 7 nerve test (NIS+7), deteriorated by 8.7 points in the diflunisal group and 25.0 points in the placebo group ( p
