A novel type of familial transthyretin amyloidosis,ATTR Asn124Ser,with co-localization of κ light chains

A 68-year-old Italian woman who had a clinical history of thyroidectomy in 2002 presented with slowly progressing renal insuffiency and non-nephrotic proteinurea in 2004. A renal biopsy showed the occurrence of amyloid; the thyroid biopsy previously taken also revealed amyloid infiltration. Other amyloid-containing tissues included bone marrow and heart. The plasma cell level in the bone marrow was found to be less than 5% and both serum and urine samples were positive for a monoclonal κ light chain band. DNA analysis unexpectedly revealed the presence of a novel transthyretin (TTR) mutation, ATTR Asn124Ser. Histologically, amyloid deposits in the thyroid had a homogeneous appearance with moderate Congophilia. In immunohistochemistry, a κ light chain antiserum showed positive immunoreactivity with amyloid deposits in the thyroid. Furthermore, a TTR antiserum, anti-TTR50-127, also recognized a number of amyloid deposits stained positive with the κ light chain antiserum. Overall, the κ light chain antiserum reacted with most of the amyloid deposits in the thyroid, whereas TTR immunoreactivity was scarcer, with a scattered appearance. In contrast, only the anti-TTR50-127 antiserum labeled amyloid in the kidney, albeit not all deposits. In this study, we report a patient having a novel TTR variant, ATTR Asn124Ser, with co-localization of κ light chains in the amyloid deposits in the thyroid tissue.     

One mutation,two distinct disease variants: unravelling the impact of transthyretin amyloid fibril composition

Although hereditary transthyretin (h‐ATTR) amyloidosis is a monogenetic disease, a large variation in its phenotype has been observed. The common hypothesis of amyloid fibril formation involves dissociation of the transthyretin (TTR) tetramer into monomers that after misfolding reassemble into amyloid fibrils. This notion is partly challenged by the finding of two distinct types of amyloid fibrils. One of these, type A, consists of C‐terminal ATTR fragments and full‐length TTR, whereas the other, type B, consists only of full‐length TTR. All organs of an individual patient contain ATTR deposits of either type A or type B fibrils, and the composition in each individual remains unchanged over time. The finding of two distinct types of ATTR fibrils suggests that there are at least two different pathways in operation for ATTR fibril formation. For the most common European mutation, TTR Val30Met, ATTR fibril composition is related to the outcome of liver transplantation, which is the first successful treatment for the disease, and the penetrance of the trait. In addition, the presence of C‐terminal ATTR fragments has an impact on the affinity for various tracers used for noninvasive imaging of amyloid depositions such as 99 m‐technetium‐diphosphono‐propanodicarboxylic acid scintigraphy and positron emission tomography utilizing Pittsburgh component B, and even for the gold standard diagnostic procedure, tissue biopsy stained by Congo red and examined under polarized light. The importance of amyloid fibril composition needs to be taken into consideration when designing clinical trials of treatment modalities, and also in the evaluation of diagnostic methods such as imaging techniques.     

Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy.

A matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry (MS) system was used to detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.     

Usefulness of MALDUTOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy

A matrix-assisted laser desorption ionization/time-of flight (MALDI/TOF) mass spectrometry (MS) system was used io detect variant transthyretin (TTR) in immunoprecipitated serum TTR molecules obtained from 6 patients with familial amyloid polyneuropathy (FAP) who were already proven not to have ATTR Val 30Met. This simple and quick method showed six different patterns of mass spectra of TTR-related immunoprecipitates from these patients, and in each patient the clearly identified characteristic doublet-shaped ion peaks consisted of normal and variant TTR apart from each other peak with a mass difference between them. DNA sequencing confirmed that the patterns of variant TTR corresponded respectively to ATTR Val30Leu, ATTR Phe33Val, ATTR Asp38Ala, ATTR Ser50Arg, ATTR Ala97Gly and ATTR Ala97Ser. ATTR Asp38Ala and ATTR Ala97Ser are previously unknown variants of TTR leading to the development of FAP. ATTR Phe33Val was found in a Chinese FAP patient and ATTR Ala97Ser in a Taiwanese. Serum analysis using immunoprecipitation and MALDI/TOF MS system can provide useful information when investigating FAP patients with diverse types of variant TTR.     

Cardiomyopathy in Swedish patients with the Gly53Glu and His88Arg transthyretin variants.

We report two new amyloidogenic transthyretin (TTR) variants detected in the Swedish population. One variant was previously unknown, while the other has been described in a French family. In Swedish patients, both variants have caused late-onset cardiac amyloidosis characterised by heart failure. In both cases, the diagnosis was determined by the detection of amyloid deposits in skin and/or rectal biopsies and identification of TTR mutations by genetic analysis. The index case of the previously unknown mutation (ATTR His88Arg) was a 66-year-old Swedish man, who sought medical attention for increasing dyspnea. Echocardiographic examination disclosed a restrictive cardiomyopathy, and subsequent examinations disclosed TTR amyloidosis. The patient is alive with moderate symptoms one year after the onset of disease. The index case for the new Swedish mutation (ATTR Gly53Glu) is a woman who sought medical attention at the age of 57 because of increasing dyspnea. Echocardiographic examination disclosed a hypertrophic cardiomyopathy with diastolic impairment. The diagnosis of systemic amyloidosis was made by fat aspiration biopsy and histopathology. The patient developed severe intractable heart failure, with pulmonary effusion and ascites. She died four years after the onset of her disease of intractable heart and kidney failure. Post mortem examination of biopsy specimens and blood revealed TTR amyloid deposits and the ATTR Gly53Glu mutation was detected.     

Diagnosis and treatment of transthyretin-related amyloidosis cardiomyopathy

Transthyretin-related amyloidosis (ATTR) is a subgroup of amyloidosis that results from extracellular misassembled and toxic amyloid deposits affecting multiple organ systems, and cardiac tissues in particular. Because ATTR often presents as heart failure with preserved ejection fraction (HFpEF), it has been largely underdiagnosed. Once considered incurable with a grave prognosis, ATTR cardiomyopathy has seen the development of promising alternatives for diagnosis and treatment, with early diagnosis and treatment of ATTR cardiomyopathy highly beneficial due to its high mortality rate. For instance, diagnosing ATTR cardiomyopathy previously required a cardiac biopsy, but new modalities, such as cardiac magnetic resonance imaging and radionuclide bone scans, show promise in accurately diagnosing ATTR cardiomyopathy. Ongoing research and clinical trials have focused on identifying new treatments which primarily target amyloid fiber formation by inhibiting TTR gene expression, stabilizing the TTR tetramer, preventing oligomer aggregation, or affecting degradation of amyloid fibers. In this review, we describe the advances made in the diagnosis and treatment of ATTR in order to increase awareness of the disease and encourage a lower threshold for ATTR workup. Our review also highlights the need for improving the screening, diagnosis, and treatment guidelines for ATTR cardiomyopathy.     

增生性肾小球肾炎伴晶格状结构的单克隆IgG沉积

51岁男性,病程1月,以高血压起病,肾脏损害主要表现大量蛋白尿,低白蛋白血症,伴少量镜下血尿,肾功能异常。肾外表现有轻度正细胞正色素性贫血,血清免疫固定电泳提示κ型IgG单克隆免疫球蛋白条带,骨髓活检和骨髓细胞学检查均阴性。肾活检组织学改变为肾小球系膜细胞、内皮细胞增生,毛细血管袢内较多CD68+细胞浸润,肾小球基膜内皮下大量、少量系膜区、偶见上皮侧嗜复红物沉积,沉积物免疫荧光染色仅IgG1和κ轻链阳性,电镜观察沉积物具有晶格状结构,免疫电镜证实这些晶格状的物质IgG和κ轻链阳性。该患者最终诊断为增生性肾小球肾炎伴具有晶格状结构的单克隆IgG沉积。     

Electron and immuno-electron microscopy of abdominal fat identifies and characterizes amyloid fibrils in suspected cardiac amyloidosis.

We evaluated the role of electron microscopy and immuno-electron microscopy studies on abdominal fat fine-needle biopsy samples in diagnosis and characterization of cardiac amyloidosis. The series consists of 15 patients with echocardiographic evidence of "restrictive cardiomyopathy" suspected to be due to amyloidosis. Patients underwent: clinical examination, electrocardiography, 2-D and Doppler echocardiography, immunofixation of serum and urine for detection of monoclonal immunoglobulins, and abdominalfat biopsies that were investigated with polarized light (Congo red), electron and immuno-electron microscopy using specific antibodies to kappa and lambda light chains, apolipoprotein A1, serum amyloid A (SAA), and transthyretin (TTR). Ultrastructural study of abdominal fat samples identified amyloid deposits in 15/15 cases. Immuno-electron microscopy specifically stained amyloid fibrils with antibodies anti-lambda (n = 8), -kappa (n = 2), -apolipoprotein A1 (n = 2) and -TTR (n = 3). Immuno-electron microscopy revealed TTR immuno-labelling in 2 patients with accidental monoclonal components, and a A reaction in I patient without monoclonal components. TTR and apolipoprotein A1 positive cases carried missense mutations in the corresponding genes. Our results demonstrate that amyloid deposits are present in the abdominalfat of patients suspected to have cardiac amyloidosis and that immuno-electron microscopy was able to characterize the amyloid protein in all cases.     

Aging and transthyretin-related amyloidosis: pathologic examinations in pulmonary amyloidosis.

Although aging is closely related with the onset of senile systemic amyloidosis (SSA) caused by wild-type transthyretin (TTR), the effect of aging on amyloid formation has remained unclear in familial amyloidotic polyneuropathy (FAP), caused by variant- and wild-type TTR. The aim of this study was to elucidate the effects of aging and/or other factors in FAP on amyloid formation in the lung, one of the most important target organs of amyloid deposition in SSA. Pulmonary amyloid distribution was determined using 19 autopsied lung samples from patients with FAP amyloidogenic TTR (ATTR) V30M, the most common type of FAP. Amyloid deposition was observed around the walls of the bronchi/ bronchioles, the pulmonary arteries, and the pulmonary veins, while no amyloid deposits could be found around the lymphatics. In addition, amyloid deposition in the alveolar regions was a characteristic finding in aged patients with FAP ATTR V30M (average ages of the patients with amyloid positive vs. negative: 50.55 +/- 8.75 vs. 39.75 +/- 4.17 years old, p < 0.005), similar to the finding in one SSA patient. These results suggest that aging could play an important role in the progression of pulmonary amyloid formation in FAP ATTR V30M.     

Aging and transthyretin-related amyloidosis: Pathologic examinations in pulmonary amyloidosis

Although aging is closely related with the onset of senile systemic amyloidosis (SSA) caused by wild-type transthyretin (TTR), the effect of aging on amyloid formation has remained unclear in familial amyloidotic polyneuropathy (FAP), caused by variant- and wild-type TTR. The aim of this study was to elucidate the effects of aging and/or other factors in FAP on amyloid formation in the lung, one of the most important target organs of amyloid deposition in SSA. Pulmonary amyloid distribution was determined using 19 autopsied lung samples from patients with FAP amyloidogenic TTR (ATTR) V30M, the most common type of FAP. Amyloid deposition was observed around the walls of the bronchi/ bronchioles, the pulmonary arteries, and the pulmonary veins, while no amyloid deposits could be found around the lymphatics. In addition, amyloid deposition in the alveolar regions was a characteristic finding in aged patients with FAP ATTR V30M (average ages of the patients with amyloid positive vs. negative: 50.55 ± 8.75 vs. 39.75 ± 4.17 years old, p < 0.005), similar to the finding in one SSA patient. These results suggest that aging could play an important role in the progression of pulmonary amyloid formation in FAP ATTR V30M.     

Early cardiac involvement in senile systemic amyloidosis: a case report.

Senile systemic amyloidosis (SSA) is caused by the deposition of wild-type transthyretin (TTR)-derived amyloid fibrils. This type of amyloidosis is not rare in elderly individuals over the age of 80 and is usually detected on postmortem microscopic examination of myocardium. We report a 67-year-old male patient who was clinically diagnosed as having SSA with cardiac involvement. The initial event was cerebral infarction conceivably due to cardiac embolism. Endomyocardial biopsy was performed twice, which led to a definitive diagnosis of amyloidosis at the second biopsy. This amyloid was immunolabeled by an anti-TTR antibody and direct DNA sequencing of the TTR gene did not detect any mutation. Clinical confirmation of SSA in individuals before the age of 70 is infrequent and cardiac amyloidosis associated with this disease might have been the cause of cerebral embolism in our patient. Additionally, it is important to distinguish cardiac amyloidosis of SSA from that of primary immunoglobulin light chain (AL)-derived amyloidosis, because the treatment and prognosis differ considerably from those of primary AL amyloidosis.     

Diagnostic value of abdominal wall fat pad biopsy in senile systemic amyloidosis

Senile systemic amyloidosis (SSA) is a main cause of intractable heart failure in elderly individuals. To demonstrate transthyretin (TTR)-derived amyloid deposition endomyocardial biopsy has been commonly carried out in the patients with SSA, but this invasive biopsy technique cannot always be performed in aged patients with severe cardiac dysfunction. During the past 3 years, 11 patients with SSA (6 males and 5 females; ages from 70 to 97 years) were examined. All underwent skin biopsy from the abdominal wall and 8 showed TTR-immunoreactive amyloid deposition (sensitivity: 73%): amyloid deposits were seen mainly in the deep layer of subcutaneous fat tissue and showed a patchy distribution. They were weakly Congophilic, but were strongly immunolabeled by an anti-TTR antibody. The severity and pattern of amyloid deposition in this biopsy of SSA patients were considerably different from those obtained from age-matched patients with TTR-related familial amyloid polyneuropathy. Surgical skin biopsy including the deep subcutaneous fat pad can be performed safely at the bedside and is useful for the histopathological diagnosis of SSA.     

Biochemical characterisation of amyloid by endomyocardial biopsy

Cardiomyopathy is a major cause of death in patients with systemic amyloidosis. There are several forms of systemic amyloidosis which cause cardiomyopathy and determination of the exact type of amyloid in each affected patient is essential for treatment and determination of prognosis. In this study, we tested the feasibility of determining the type of amyloidosis by biochemical analysis of endomyocardial biopsies.

Right ventricular endomyocardial biopsies were obtained from 10 patients with restrictive cardiomyopathy. Three patients had monoclonal protein demonstrated in serum or urine and all three had bone marrow findings consistent with monoclonal gammopathy. Seven patients had isolated cardiomyopathy without evidence of monoclonal gammopathy. A portion of each myocardial biopsy was submitted for histologic evaluation and all demonstrated amyloid by Congo red staining. Each biopsy was analysed biochemically by isolation of amyloid fibrils and the protein characterised by amino acid sequence analysis. Four amyloid isolates were characterised as immunoglobulin light chain proteins. Two specimens obtained from patients with transthyretin (TTR) DNA mutations contained TTR peptides proving the hereditary nature of the disease. Biopsies from four patients without a TTR mutation contained TTR and were consistent with the diagnosis of senile cardiac amyloidosis (SCA).

All endomyocardial biopsy specimens that were analysed had sufficient amyloid fibril subunit protein to allow characterisation by amino acid sequence analysis. This methodology is particularly useful in differentiating SCA with TTR amyloid fibrils from immunoglobulin light chain amyloidosis which also occurs in the elderly age group.     

Cardiac amyloidosis: An update on pathophysiology,diagnosis, and treatment

The amyloidoses are a group of systemic diseases characterized by organ deposition of misfolded protein fragments of diverse origins. The natural history of the disease, involvement of other organs, and treatment options vary significantly based on the protein of origin. In AL amyloidosis, amyloid protein is derived from immunoglobulin light chains, and most often involves the kidneys and the heart. ATTR amyloidosis is categorized as mutant or wild-type depending on the genetic sequence of the transthyretin (TTR) protein produced by the liver. Wild-type ATTR amyloidosis mainly involves the heart, although the reported occurrence of bilateral carpal tunnel syndrome, spinal stenosis and biceps tendon rupture in these patients speaks to more generalized protein deposition. Mutant TTR is marked by cardiac and/or peripheral nervous system involvement. Cardiac involvement is associated with symptoms of heart failure, and dictates the clinical course of the disease. Cardiac amyloidosis can be diagnosed noninvasively by echocardiography, cardiac MRI, or nuclear scintigraphy. Endomyocardial biopsy may be needed in the case of equivocal imaging findings or discordant data. Treatment is aimed at relieving congestive symptoms and targeting the underlying amyloidogenic process. This includes anti-plasma cell therapy in AL amyloidosis, and stabilization of the TTR tetramer or inhibition of TTR protein production in ATTR amyloidosis. Cardiac transplantation can be considered in highly selected patients in tandem with therapy aimed at suppressing the amyloidogenic process, and appears associated with durable long-term survival.     

Cardiac amyloidosis: An underdiagnosed/underappreciated disease

Cardiac amyloidosis or amyloid cardiomyopathy (ACM), commonly resulting from extracellular deposition of amyloid fibrils consisted of misfolded immunoglobulin light chain (AL) or transthyretin (TTR) protein, is an underestimated cause of heart failure and cardiac arrhythmias. Among the three types of cardiac amyloidosis (wild-type or familial TTR and light-chain), the wild-type (Wt) TTR-related amyloidosis (ATTR) is an increasingly recognized cause of heart failure with preserved ejection fraction (HFpEF), and amyloidosis should be considered in the differential diagnosis of this heart failure group of patients. Recent advances in the diagnosis and drug treatment of ACM have ushered in a new era in early disease detection and better management of these patients. Certain clues in cardiac and extracardiac manifestations of ACM may heighten clinical suspicion and guide further confirmatory testing. Newer noninvasive imaging methods (strain echocardiography, cardiac magnetic resonance and bone scintigraphy) may obviate the need for endomyocardial biopsy in ATTR patients, while newer targeted therapies may alter the adverse prognosis in these patients. Early recognition of ACM is crucial in halting the disease process before irreversible organ damage occurs. Chemotherapy and stem-cell transplantation combined with immunomodulatory therapy may also favorably affect the course and prognosis of light chain ACM. Finally, in select patients with end-stage disease, heart transplantation may render results comparable to non-ACM patients. All these issues are herein reviewed.     

Analysis of transthyretin amyloid fibrils from vitreous samples in familial amyloidotic polyneuropathy (Val30Met).

The aim of the present study was to analyze the forms of wild type and mutated monomeric transthyretin (Val30Met) in the amyloid fibrils of patients with familial amyloidotic polyneuropathy by electrospray ionization mass spectrometry (ESI-MS). The solubility of amyloid fibrils from the vitrectomized samples was examined to determine the appropriate solution for ESI-MS. ESI-MS analysis revealed that heterozygotic Val30Met amyloid fibrils contained 14.6 +/- 7.5% normal TTR. In all samples, 3 different types of variant ATTR could be identified: Full length ATTR, and -57, and -157 (or 156) Da from ATTR Val30Met were found. The two peaks showing -57, and -157 (or 156) Da from ATTR Val30Met corresponded to the -Gly, and -Gly-Pro sequences of ATTR Val30Met from the N-terminal. The results illustrate the heterogeneity of ATTR amyloid deposits and this method may be very useful for analyzing amyloid fibrils in ATTR related amyloidosis.     

A rare transthyretin mutation (Asp18Glu) associated with cardiomyopathy

The identification of a rare transthyretin (TTR) gene mutation (Asp18Glu) in a middle-aged male with biopsy proven amyloid disease featuring cardiomyopathy is described. The more commonly occurring light chain amyloidosis (AL) was initially considered, but negative hematologic testing prompted screening for a pathologic TTR mutation. A differential diagnosis of familial transthyretin type amyloidosis (ATTR) was established using a combination of molecular genetic and biochemical techniques. Single-strand conformation polymorphism (SSCP) screening of exons 2, 3 and 4 of the TTR gene indicated the presence of atypical DNA. SSCP testing was performed using a new non-radioactive, silver stained minigel technique. The genetic abnormality was identified by direct DNA sequence analysis as a T to A transversion at the third base position in codon 18. This result was confirmed by restriction fragment length polymorphism (RFLP) testing. The presence of the variant protein, TTR Asp18Glu, in serum from the proband was confirmed by mass spectrometric analysis.     

A rare transthyretin mutation (Asp18Glu) associated with cardiomyopathy.

The identification of a rare transthyretin (TTR) gene mutation (Asp18Glu) in a middle-aged male with biopsy proven amyloid disease featuring cardiomyopathy is described. The more commonly occurring light chain amyloidosis (AL) was initially considered, but negative hematologic testing prompted screening for a pathologic TTR mutation. A differential diagnosis of familial transthyretin type amyloidosis (ATTR) was established using a combination of molecular genetic and biochemical techniques. Single-strand conformation polymorphism (SSCP) screening of exons 2, 3 and 4 of the TTR gene indicated the presence of atypical DNA. SSCP testing was performed using a new non-radioactive, silver stained minigel technique. The genetic abnormality was identified by direct DNA sequence analysis as a T to A transversion at the third base position in codon 18. This result was confirmed by restriction fragment length polymorphism (RFLP) testing. The presence of the variant protein, TTR Asp18Glu, in serum from the proband was confirmed by mass spectrometric analysis.     

Transthyretin familial amyloidotic polyneuropathy: histopathological study of the explanted livers

FAP is an autosomal dominant inherited disease, characterized by systemic deposition of amyloid fibrils in various tissues. The purpose of this study is to describe the gross and microscopic findings of the explanted livers for FAP.10 patients were transplanted for FAP at our institution. Diagnosis was supported by positive familiar history, clinical data and detection of mutated TTR by electrospray ionization mass spectrometry with Val30Met mutation verified by PCR. All the explanted livers were photographed, fixed in formol and processed according to protocol. Later they were examined with HyE, reticulin, PAS diastasa, Masson trichromic, Congo red with polarised light and immunoreactivity against TTR. The gross aspect was normal. We obtained multiple samples representative of the organ and the hepatic hilium. All of the patients presented with deposits of amyloid substance in the lymph nodes and the nerves of the hepatic hilium These deposits were Congo red positive with a greenish birefringence to polarized light Deposits show immunoreactivity with antihuman TTR. Whereas liver transplantation restores hepatic function in patients with cirrhosis, liver transplantation cures the FAP patient of their genetic defect. Domino transplantation is a procedure in which the index patient receives an organ, while the explanted organ is reused for transplantation into another patient. In conclusion, exclusion of hepatic amyloid deposits which can cause functional alterations in the FAP liver is vital; and is important to study the explanted livers of patients with FAP to confirm the results of the scarce published series.     

Deposition of transthyretin amyloid is not accelerated by the same amyloid in vivo

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported in several types of amyloidoses. Families with a variant transthyretin (TTR V30M)-associated familial amyloidotic polyneuropathy (FAP) exhibit genetic anticipation, with TTR V30M-amyloid depositing at an earlier age in successive generations. The molecular bases of anticipation in FAP have remained to be determined. We asked if administration of TTR-amyloid fibrils (ATTR) extracted from the heart of an FAP TTR V30M patient would accelerate ATTR deposition in transgenic mice expressing the human mutant ttr gene responsible for FAP TTR V30M and indeed the administration did accelerate deposition of apolipoprotein A-II- amyloid fibrils (AApoAII), and not ATTR. Our experiments present, for the first time, evidence that the degree of inducibility of ATTR is low relative to AApoAII and we suggest that administration of ATTR may not explain the genetic anticipation which occurs in FAP.