Estudio descriptivo de la amiloidosis por transtiretina en un hospital de tercer nivel sin unidad de referencia / A descriptive study of transthyretin amyloidosis in a tertiary hospital without a referral unit

Antecedentes y objetivo: La amiloidosis por transtiretina (ATTR) es una enfermedad rara que forma parte de las amiloidosis sistémicas, y es una enfermedad amenazante para la vida. Puede afectar a todos los órganos y sistemas, siendo la más frecuente la afectación neurológica y cardíaca. El objetivo de este estudio es detectar posibles casos de ATTR y realizar un estudio descriptivo de los mismos. Material y métodos: Estudio descriptivo unicéntrico realizado en un hospital de tercer nivel en el que se incluyen pacientes con sospecha de ATTR entre septiembre de 2016 y enero de 2020. Resultados: Se detectan 190 pacientes sospechosos de ATTR. En el estudio se incluyen 100 de ellos, así como 10 familiares de pacientes en los que se detecta ATTR en su variante genética. En total, se detecta ATTR variante genética en 7 individuos (3 con mutación presintomática de la enfermedad), 16 pacientes con ATTR asociada a la edad y 31 individuos con amiloidosis cardíaca no filiada con las pruebas realizadas, lo que confirma la presencia de esta enfermedad en áreas no endémicas. Conclusiones: La ATTR es una enfermedad que se ha de tener en cuenta en el diagnóstico diferencial de pacientes que presentan insuficiencia cardíaca con FEVI preservada, principalmente si se asocia a síntomas neurológicos (AU)

Background and objective: Transthyretin amyloidosis (ATTR) is a rare disease that is part of systemic amyloidosis and is life-threatening. It can affect all organs and systems, the most frequent being neurological and cardiac involvement. This study aims to detect possible ATTR cases and carry out a descriptive study of them. Material and methods: Descriptive single-centre study carried out in a tertiary hospital, which included patients with suspected ATTR between September 2016 and January 2020. Results: A total of 190 suspected ATTR patients were detected. The study includes 100 of these patients, as well as 10 relatives of patients in whom ATTR was detected in its genetic variant (ATTRv). In total, ATTRv was detected in 7 individuals (3 with a presymptomatic mutation of the disease), 16 patients with age-related ATTR and 31 individuals with unknown cardiac amyloidosis with the tests performed, which confirms the presence of this disease in non-endemic areas. Conclusions: ATTR is a disease that must be taken into account in the differential diagnosis of patients with heart failure with preserved LVEF, especially if associated with neurological symptoms (AU)

Ocular Involvement in Hereditary Amyloidosis.

The term amyloidosis describes a group of rare diseases caused by protein conformation abnormalities resulting in extracellular deposition and accumulation of insoluble fibrillar aggregates. So far, 36 amyloid precursor proteins have been identified, and each one is responsible for a specific disease entity. Transthyretin amyloidosis (ATTRv) is one of the most common forms of systemic and ocular amyloidosis, due to the deposition of transthyretin (TTR), which is a transport protein mainly synthesized in the liver but also in the retinal pigment epithelial cells. ATTRv amyloidosis may be misdiagnosed with several other conditions, resulting in a significant diagnostic delay. Gelsolin and keratoepithelin are other proteins that, when mutated, are responsible for a systemic amyloid disease with significant ocular manifestations that not infrequently appear before systemic involvement. The main signs of ocular amyloid deposition are in the cornea, irido-corneal angle and vitreous, causing complications related to vasculopathy and neuropathy at the local level. This review aims at describing the main biochemical, histopathological and clinical features of systemic amyloidosis associated with eye involvement, with particular emphasis on the inherited forms. We discuss currently available treatments, focusing on ocular involvement and specific ophthalmologic management and highlighting the importance of a prompt treatment for the potential sight-threatening complications derived from amyloid deposition in ocular tissues.

Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits.

The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.

Soluble TREM-1 Levels in Familial Mediterranean Fever Related AA-Amyloidosis.

Objectives: Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) is a monocyte and neutrophil receptor functioning in innate immunity. TREM-1 activity has been studied in various autoimmune diseases such as RA and SLE but there is no data in autoinflammatory pathologies. We studied soluble TREM-1 (sTREM-1) activity in Familial Mediterranean Fever (FMF) cases to evaluate the clinical role of TREM-1 in amyloidosis. Methods: The study includes 62 patients with FMF (42 with amyloidosis) who are regular attendees of a tertiary center for autoinflammatory diseases. For control purposes, 5 patients with AA amyloidosis secondary to other inflammatory diseases, and 20 healthy individuals were also included. Soluble TREM-1 levels were measured using enzyme-linked immunosorbent assay (ELISA). All FMF patients were in an attack-free period during the collection of the blood samples.Results: Soluble TREM-1 levels were found to be significantly higher in the FMF amyloidosis group compared to FMF without amyloidosis group and healthy controls (p = .001 and 0.002). Nevertheless, this difference between sTREM-1 levels was not found among FMF amyloidosis and other AA amyloidosis groups (p = .447) as well as between only FMF patients and healthy controls (p = .532). Soluble TREM-1 levels were found in correlation with creatinine and CRP in the FMF patient group regardless of their amyloidosis diagnosis (r = 0.314, p = .013; r = 0.846, p < .001).Conclusion: TREM-1 seems to be related to renal function rather than disease activity in FMF. Its role as an early diagnostic marker of amyloidosis in FMF complicated with AA amyloidosis should be tested in larger patient groups.

Fibrillar conformation of an apolipoprotein A-I variant involved in amyloidosis and atherosclerosis.

BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.

Nanobody interaction unveils structure, dynamics and proteotoxicity of the Finnish-type amyloidogenic gelsolin variant.

AGel amyloidosis, formerly known as familial amyloidosis of the Finnish-type, is caused by pathological aggregation of proteolytic fragments of plasma gelsolin. So far, four mutations in the gelsolin gene have been reported as responsible for the disease. Although D187N is the first identified variant and the best characterized, its structure has been hitherto elusive. Exploiting a recently-developed nanobody targeting gelsolin, we were able to stabilize the G2 domain of the D187N protein and obtained, for the first time, its high-resolution crystal structure. In the nanobody-stabilized conformation, the main effect of the D187N substitution is the impairment of the calcium binding capability, leading to a destabilization of the C-terminal tail of G2. However, molecular dynamics simulations show that in the absence of the nanobody, D187N-mutated G2 further misfolds, ultimately exposing its hydrophobic core and the furin cleavage site. The nanobody's protective effect is based on the enhancement of the thermodynamic stability of different G2 mutants (D187N, G167R and N184K). In particular, the nanobody reduces the flexibility of dynamic stretches, and most notably decreases the conformational entropy of the C-terminal tail, otherwise stabilized by the presence of the Ca2+ ion. A Caenorhabditis elegans-based assay was also applied to quantify the proteotoxic potential of the mutants and determine whether nanobody stabilization translates into a biologically relevant effect. Successful protection from G2 toxicity in vivo points to the use of C. elegans as a tool for investigating the mechanisms underlying AGel amyloidosis and rapidly screen new therapeutics.

Effects of Disease-Causing Mutations on the Conformation of Human Apolipoprotein A-I in Model Lipoproteins.

Plasma high-density lipoproteins (HDLs) are protein-lipid nanoparticles that transport lipids and protect against atherosclerosis. Human apolipoprotein A-I (apoA-I) is the principal HDL protein whose mutations can cause either aberrant lipid metabolism or amyloid disease. Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) was used to study the apoA-I conformation in model discoidal lipoproteins similar in size to large plasma HDL. We examined how point mutations associated with hereditary amyloidosis (F71Y and L170P) or atherosclerosis (L159R) influence the local apoA-I conformation in model lipoproteins. Unlike other apoA-I forms, the large particles showed minimal conformational heterogeneity, suggesting a fully extended protein conformation. Mutation-induced structural perturbations in lipid-bound protein were attenuated compared to the free protein and indicated close coupling between the two belt-forming apoA-I molecules. These perturbations propagated to distant lipoprotein sites, either increasing or decreasing their protection. This HDX MS study of large model HDL, compared with previous studies of smaller particles, ascertained that apoA-I's central region helps accommodate the protein conformation to lipoproteins of various sizes. This study also reveals that the effects of mutations on lipoprotein conformational dynamics are much weaker than those in a lipid-free protein. Interestingly, the mutation-induced perturbations propagate to distant sites nearly 10 nm away and alter their protection in ways that cannot be predicted from the lipoprotein structure and stability. We propose that long-range mutational effects are mediated by both protein and lipid and can influence lipoprotein functionality.

Diet Supplementation with Hydroxytyrosol Ameliorates Brain Pathology and Restores Cognitive Functions in a Mouse Model of Amyloid-ß Deposition.

Alzheimer's disease is the most common form of dementia affecting a large proportion of aged people. Plant polyphenols have been reported to be potentially useful in the prevention of AD due to their multiple pharmacological activities. The aim of the present study was to assess whether the previously reported neuroprotective and anti-inflammatory effects resulting from oleuropein aglycone administration were reproduced by diet supplementation with similar amounts of its metabolite hydoxytyrosol (HT). Four-month-old TgCRND8 and wild type mice were treated for 8 weeks with a low-fat diet (5%) supplemented with HT (50 mg/kg of diet). We found that HT supplementation significantly improved cognitive functions of TgCRND8 mice and significantly reduced Aß42 and pE3-Aß plaque area and number in the cortex; in the hippocampal areas of HT-fed TgCRND8 mice, we found a significant reduction in the pE3-Aß plaque number together with a tendency toward a reduction in Aß42 load and pE3-Aß plaque area, associated with a marked reduction of TNF-α expression and astrocyte reaction. Macroautophagy induction and modulation of MAPKs signaling were found to underlie the beneficial effects of HT. Our findings indicate that HT administration reproduces substantially the beneficial effects on behavioral performance and neuropathology previously reported in TgCRND8 mice fed with oleuropein aglycone, resulting in comparable neuroprotection.

Effects of iron on the aggregation propensity of the N-terminal fibrillogenic polypeptide of human apolipoprotein A-I.

Specific mutations in APOA1 gene lead to systemic, hereditary amyloidoses. In ApoA-I related amyloidosis involving the heart, amyloid deposits are mainly constituted by the 93-residue N-terminal region of the protein, here indicated as [1-93]ApoA-I. Oxidative stress is known to be an enhancing factor for protein aggregation. In healthy conditions, humans are able to counteract the formation and the effects of oxidative molecules. However, aging and atmospheric pollution increase the concentration of oxidative agents, such as metal ions. As the main effect of iron deregulation is proposed to be an increase in oxidative stress, we analysed the effects of iron on [1-93]ApoA-I aggregation. By using different biochemical approaches, we demonstrated that Fe(II) is able to reduce the formation of [1-93]ApoA-I fibrillar species, probably by stabilizing its monomeric form, whereas Fe(III) shows a positive effect on polypeptide fibrillogenesis. We hypothesize that, in healthy conditions, Fe(III) is reduced by the organism to Fe(II), thus inhibiting amyloid formation, whereas during ageing such protective mechanisms decline, thus exposing the organism to higher oxidative stress levels, which are also related to an increase in Fe(III). This alteration could contribute to the pathogenesis of amyloidosis.

Primary adrenal insufficiency due to hereditary apolipoprotein AI amyloidosis: endocrine involvement beyond hypogonadism.

Several mutations in the gene encoding apolipoprotein AI (apoAI) have been described as a cause of familial amyloidosis. Individuals with apoAI-derived (AApoAI) amyloidosis frequently manifest with liver, kidney, laryngeal, skin and myocardial involvement. Although primary hypogonadism (PH) is considered almost pathognomonic of this disease, until now, primary adrenal insufficiency (PAI) has not been described as a common clinical feature. Here, we report the first kindred with AApoAI amyloidosis in which PAI is well-documented. All family members with the Leu60_Phe71delins60Val_61Thr heterozygous mutation who were regularly followed-up at our centre were considered. Nineteen individuals had the confirmed APOA1 deletion/insertion mutation, with detailed medical records available in 11 cases. Of these, 6 had PAI and 3 (all males) had PH. Among them, one 47-year-old man, not previously diagnosed with PAI, developed adrenal crisis after liver transplantation, precipitated by an opportunistic infection. Transplantation due to organ failure, which necessitates use of immunosuppressive medication such as corticosteroids, is frequently required during the course of hereditary amyloidosis. Consequently, PAI can remain masked, being discovered only when an adrenal crisis develops. Therefore, according to the present evidence, patients with AApoAI amyloidosis should be submitted to regular testing of corticotrophin and cortisol levels in order to avoid delaying corticosteroid replacement.

A mouse model of a novel missense mutation (Gly83Arg) in a Chinese kindred manifesting vitreous amyloidosis only.

The purpose of this study is to establish a mouse model of transthyretin (TTR) Gly83Arg gene mutation by the technique of gene targeting for research on hereditary vitreous amyloidosis (HVA) and to confirm whether this point mutation is a genetic feature of HVA. A vector (pBR322-MK-TTR) was constructed to target ES cells. The successfully transfected ES cells were used for blastocyst injection, thus generating F0. F0 and Flp mice were mated to generate F1 (TTR+/-, Flp +/-) mice that lacked the neo gene but carried the Flp gene. F1 mice were mated with C57BL/6N wild type mice to generate F2 (TTR+/-) mice. F3 homozygous and heterozygous mice were generated by mating F2 mice with each other. PCR and sequencing were performed for F3 mice. Amyloid was detected using Congo red stain and polarized light. Immunohistochemistry was used to detect the expression of TTR in the tissues. Quantitative fluorescent PCR and Western blotting were used to detect the expression of TTR mRNA and TTR protein, respectively. Two F0-generation, 2 F1-generation and 15 F3-generation mice were obtained. The gene sequencing of F3 mice showed TTR Gly83Arg mutation. When examined with Congo red and polarized light, the vitreous of TTR Gly83Arg mutant mice tested positive for amyloid. The hearts, livers, brains and kidneys of the experimental group and control group were all negative by Congo red staining. Immunohistochemical staining showed that the vitreous of TTR Gly83Arg mutant mice and the livers of the control mice were positive, but the kidneys, hearts and brains of both groups were negative. Quantitative fluorescent PCR showed that the mRNA expression of mutant mice was significantly lower than that of wild-type mice (F = 0.295, P = 0.023). Western blotting showed that the expression of TTR protein was significantly lower in the model mice than in the wild-type mice (t = 3.224, P = 0.018). TTR gene mutation is indeed a molecular characteristic of HVA and manifest in the eye disease only. A C57BL/6 mouse line carrying the TTR Gly83Arg gene mutation was successfully established. This strain of mice can be used for the study of HVA.

Cell milieu significantly affects the fate of AApoAI amyloidogenic variants: predestination or serendipity?

BACKGROUND: Specific apolipoprotein A-I variants are associated to severe hereditary amyloidoses. The organ distribution of AApoAI amyloidosis seems to depend on the position of the mutation, since mutations in residues from 1 to 75 are mainly associated to hepatic and renal amyloidosis, while mutations in residues from 173 to 178 are mostly responsible for cardiac, laryngeal, and cutaneous amyloidosis. Molecular bases of this tissue specificity are still poorly understood, but it is increasingly emerging that protein destabilization induced by amyloidogenic mutations is neither necessary nor sufficient for amyloidosis development. METHODS: By using a multidisciplinary approach, including circular dichroism, dynamic light scattering, spectrofluorometric and atomic force microscopy analyses, the effect of target cells on the conformation and fibrillogenic pathway of the two AApoAI amyloidogenic variants AApoAIL75P and AApoAIL174S has been monitored. RESULTS: Our data show that specific cell milieus selectively affect conformation, aggregation propensity and fibrillogenesis of the two AApoAI amyloidogenic variants. CONCLUSIONS: An intriguing picture emerged indicating that defined cell contexts selectively induce fibrillogenesis of specific AApoAI variants. GENERAL SIGNIFICANCE: An innovative methodological approach, based on the use of whole intact cells to monitor the effects of cell context on AApoAI variants fibrillogenic pathway, has been set up.

Apolipoprotein A-1-related amyloidosis 2 case reports and review of the literature.

RATIONALE: Apolipoprotein A-1 (ApoA-1)-related amyloidosis is characterized by the deposition of ApoA-1 in various organs and can be either hereditary or nonhereditary. It is rare and easily misdiagnosed. Renal involvement is common in hereditary ApoA-1 amyloidosis, but rare in the nonhereditary form. PATIENT CONCERNS: We reported two cases with ApoA-1 amyloidosis, a 64-year-old man suffering from nephrotic syndrome and a 40-year-old man with nephrotic syndrome and splenomegaly. Renal biopsies revealed glomerular, interstitial and vascular amyloid deposits and positive phospholipase A2 receptor staining in the glomerular capillary loop in case 1, and mesangial amyloid deposits in case 2. DIAGNOSES: After immunostaining failed to determine the specific amyloid protein, proteomic analysis of amyloid deposits by mass spectrometry was performed and demonstrated the ApoA-1 origin of the amyloid. Genetic testing revealed no mutation of the APOA1 gene in case 1 but a heterozygous mutation, Trp74Arg, in case 2. Case 1 was thus diagnosed as nonhereditary ApoA-1 associated renal amyloidosis with membranous nephropathy, and case 2 as hereditary ApoA-1 amyloidosis with multiorgan injuries (kidney and spleen) and a positive family history. INTERVENTIONS: Case 1 was treated with glucocorticoid combined with cyclosporine. Case 2 was treated with calcitriol and angiotensin converting enzyme inhibitors. OUTCOMES: Two cases were followed up for 5 months and 2 years, respectively; and case 1 was found to have attenuated proteinuria while case 2 had an elevation of cholestasis indices along with renal insufficiency. LESSONS: Proteomic analysis by mass spectrometry of the amyloid deposits combined with genetic analysis can provide accurate diagnosis of ApoA-1 amyloidosis. Besides, these 2 cases expand our knowledge of ApoA-1-related renal amyloidosis.

Amyloid detection in the transverse carpal ligament of patients with hereditary ATTR V30M amyloidosis and carpal tunnel syndrome.

INTRODUCTION: Carpal tunnel syndrome (CTS) is a nonspecific manifestation of hereditary ATTR amyloidosis (ATTRm). Amyloid deposition of wild-type TTR (WT-ATTR) has been found in transverse carpal ligament (TCL) in idiopathic CTS. We retrospectively studied a group of patients with ATTRm and CTS submitted to carpal tunnel release surgery (CTRS). METHODS: From the nerve conduction studies performed in our Clinical Unit dedicated to hereditary amyloidosis between July 2009 and October 2013, we selected patients who fulfilled neurophysiological criteria for CTS, had been submitted to CTRS and whose TCL was available for pathology. Clinical registries were reviewed and amyloid detection in the ligaments was performed using Congo-red staining. RESULTS: We included 16 patients: three males (18.8%), mean age = 46.1 years old, all with V30M mutation. At the time of surgery, four patients were considered asymptomatic and 12 symptomatic carriers, five of them late-onset ATTRm (onset age >50 years old). In all but one patient, the CTS preceded the polyneuropathy. Amyloid detection in the TCL was positive in 14 patients (87.5%). DISCUSSION/CONCLUSIONS: In most patients, CTS preceded or was contemporary to the polyneuropathy and amyloid detection in TCL was positive. The detection of amyloid in TCL may add specificity to CTS as an early manifestation of the disease but more studies are needed.

Clinicopathological and biochemical findings of thyroid amyloid in hereditary transthyretin amyloidosis with and without liver transplantation.

Hereditary transthyretin (TTR) amyloidosis is a fatal disease causing systemic organ dysfunctions. Histopathological studies revealed that thyroid glands are major target tissues. However, details about thyroid functions remain to be fully elucidated in this disease. For patient treatment, liver transplantation (LT) reportedly prolongs patient survival, but thyroid gland function after LT still remains poorly understood. In this study, we investigated the thyroid functions in 101 patients with hereditary TTR amyloidosis and the effects of LT on thyroid functions in those patients. In addition, we investigated histopathological and biochemical findings of thyroid specimens obtained at autopsy. Disease duration and age at examination inversely correlated with serum levels of free triiodothyronine (fT3) in hereditary TTR amyloidosis. On the contrary, in patients who underwent transplantation, time from disease onset to transplantation and age at transplantation clearly correlated with serum fT3and thyroid stimulating hormone (TSH) levels. In autopsy studies, amounts of thyroid amyloid deposits in patients with transplantation were significantly lower than those in patients without transplantation. Mass spectrometric analyzes also revealed that proportions of wild-type (WT) TTR in thyroid amyloid deposits in patients with hereditary TTR amyloidosis who underwent transplantations were higher than those in patients without transplantation. Thyroid hormone functions may diminish according to the disease progression. LT could prevent thyroid dysfunction in hereditary TTR amyloidosis.