Trypsin-induced aggregation of transthyretin Valine 30 variants associated with hereditary amyloidosis.

Amyloid fibrils of transthyretin (TTR) consist of full-length TTR and C-terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C-terminal fragment remains unclear. Here, we investigated trypsin-induced aggregation and urea-induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less-bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin-induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin-induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion.

Single Nucleotide Polymorphisms Associated with AA-Amyloidosis in Siamese and Oriental Shorthair Cats.

AA-amyloidosis in Siamese and Oriental shorthair cats is a lethal condition in which amyloid deposits accumulate systemically, especially in the liver and the thyroid gland. The age at death of affected cats varies between one and seven years. A previous study indicated a complex mode of inheritance involving a major locus. In the present study, we performed a multi-locus genome-wide association study (GWAS) using five methods (mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB and ISIS EM-BLASSO) to identify variants associated with AA-amyloidosis in Siamese/Oriental cats. We genotyped 20 affected mixed Siamese/Oriental cats from a cattery and 48 healthy controls from the same breeds using the Illumina Infinium Feline 63 K iSelect DNA array. The multi-locus GWAS revealed eight significantly associated single nucleotide polymorphisms (SNPs) on FCA A1, D1, D2 and D3. The genomic regions harboring these SNPs contain 55 genes, of which 3 are associated with amyloidosis in humans or mice. One of these genes is SAA1, which encodes for a member of the Serum Amyloid A family, the precursor protein of Amyloid A, and a mutation in the promotor of this gene causes hereditary AA-amyloidosis in humans. These results provide novel knowledge regarding the complex genetic background of hereditary AA-amyloidosis in Siamese/Oriental cats and, therefore, contribute to future genomic studies of this disease in cats.

Patisiran Treatment in Patients with Transthyretin Cardiac Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is associated with accumulation of ATTR amyloid deposits in the heart and commonly manifests as progressive cardiomyopathy. Patisiran, an RNA interference therapeutic agent, inhibits the production of hepatic transthyretin. METHODS: In this phase 3, double-blind, randomized trial, we assigned patients with hereditary, also known as variant, or wild-type ATTR cardiac amyloidosis, in a 1:1 ratio, to receive patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks for 12 months. A hierarchical procedure was used to test the primary and three secondary end points. The primary end point was the change from baseline in the distance covered on the 6-minute walk test at 12 months. The first secondary end point was the change from baseline to month 12 in the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS) score (with higher scores indicating better health status). The second secondary end point was a composite of death from any cause, cardiovascular events, and change from baseline in the 6-minute walk test distance over 12 months. The third secondary end point was a composite of death from any cause, hospitalizations for any cause, and urgent heart failure visits over 12 months. RESULTS: A total of 360 patients were randomly assigned to receive patisiran (181 patients) or placebo (179 patients). At month 12, the decline in the 6-minute walk distance was lower in the patisiran group than in the placebo group (Hodges-Lehmann estimate of median difference, 14.69 m; 95% confidence interval [CI], 0.69 to 28.69; P = 0.02); the KCCQ-OS score increased in the patisiran group and declined in the placebo group (least-squares mean difference, 3.7 points; 95% CI, 0.2 to 7.2; P = 0.04). Significant benefits were not observed for the second secondary end point. Infusion-related reactions, arthralgia, and muscle spasms occurred more often among patients in the patisiran group than among those in the placebo group. CONCLUSIONS: In this trial, administration of patisiran over a period of 12 months resulted in preserved functional capacity in patients with ATTR cardiac amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO-B ClinicalTrials.gov number, NCT03997383.).

AHNAK, regulated by the OSM/OSMR signaling, involved in the development of primary localized cutaneous amyloidosis.

BACKGROUND: Primary localized cutaneous amyloidosis (PLCA) is a chronic skin disease characterized by aberrant keratinocyte differentiation, epidermal hyperproliferation, and amyloid deposits. Previously, we demonstrated OSMR loss-function mutants enhanced basal keratinocyte differentiation through the OSMR/STAT5/KLF7 signaling in PLCA patients. OBJECTIVE: To investigate the underlying mechanisms involved in basal keratinocyte proliferation in PLCA patients that remain unclear. METHODS: Patients with pathologically confirmed PLCA visiting the dermatologic outpatient clinic were involved in the study. Laser capture microdissection and mass spectrometry analysis, gene-edited mice, 3D human epidermis culture, flow cytometry, western blot, qRT-PCR and RNA sequencing were used to explore the underlying molecular mechanisms. RESULTS: In this study, we found that AHNAK peptide fragments were enriched in the lesions of PLCA patients, as detected by laser capture microdissection and mass spectrometry analysis. The upregulated expression of AHNAK was further confirmed using immunohistochemical staining. qRT-PCR and flow cytometry revealed that pre-treatment with OSM can inhibit AHNAK expression in HaCaT cells, NHEKs, and 3D human skin models, but OSMR knockout or OSMR mutations abolished this down-regulation trend. Similar results were obtained in wild-type and OSMR knockout mice. More importantly, EdU incorporation and FACS assays demonstrated the knockdown of AHNAK could induce G1 phase cell cycle arrest and inhibit keratinocyte proliferation. Furthermore, RNA sequencing revealed that AHNAK knockdown regulated keratinocyte differentiation. CONCLUSION: Taken together, these data indicated that the elevated expression of AHNAK by OSMR mutations led to hyperproliferation and overdifferentiation of keratinocytes, and the discovered mechanism might provide insights into potential therapeutic targets for PLCA.

Rational Design of a Peptidomimetic Inhibitor of Gelsolin Amyloid Aggregation.

Gelsolin amyloidosis (AGel) is characterized by multiple systemic and ophthalmic features resulting from pathological tissue deposition of the gelsolin (GSN) protein. To date, no cure is available for the treatment of any form of AGel. More than ten single-point substitutions in the GSN gene are responsible for the occurrence of the disease and, among them, D187N/Y is the most widespread variant. These substitutions undergo an aberrant proteolytic cascade, producing aggregation-prone peptides of 5 and 8 kDa, containing the Gelsolin Amyloidogenic Core, spanning residues 182-192 (GAC182-192). Following a structure-based approach, we designed and synthesized three novel sequence-specific peptidomimetics (LB-5, LB-6, and LB-7) built on a piperidine-pyrrolidine unnatural amino acid. LB-5 and LB-6, but not LB-7, efficiently inhibit the aggregation of the GAC182-192 amyloidogenic peptides at sub-stoichiometric concentrations. These peptidomimetics resulted also effective in vivo, in a C. elegans-based assay, in counteracting the proteotoxicity of aggregated GAC182-192. These data pave the way to a novel pharmacological strategy against AGel and also validate a toolbox exploitable in other amyloidogenic diseases.

Familial amyloidosis of the Finnish type: clinical and neurophysiological features of two index cases.

Familial amyloidosis of the Finnish type (FAF) is a rare multisystemic disorder caused by mutations in the gelsolin gene. The clinical presentation is typically characterised by a triad of ophthalmic, neurological and dermatological findings. FAF has been reported in several countries, primarily in Finland and recently in Portugal. We report the first genetically confirmed cases of FAF from two unrelated families in our neuromuscular outpatient clinic. Gelsolin gene sequencing revealed the heterozygous gelsolin mutation (c.640G>A). The clinical features and the neurophysiological studies of two index patients and their relatives are presented. Obtaining an early diagnosis can be challenging, but FAF should be considered in the differential diagnosis of progressive bilateral facial neuropathy, even if there is no known Finnish ancestor.

Caracterización de la amiloidosis cardiaca hereditaria por transtirretina en España / Characterization of hereditary transthyretin cardiac amyloidosis in Spain

Introducción y objetivos: La amiloidosis hereditaria por transtirretina (ATTRv) es una enfermedad causada por mutaciones en el gen de la transtirretina que frecuentemente presenta afección cardiaca debido al depósito de amiloide en el miocardio. Nuestro objetivo es describir esta afección en una cohorte española. Métodos: Estudio retrospectivo multicéntrico de pacientes con ATTRv y afección cardiaca provenientes de centros españoles. Se recogieron datos demográficos, clínicos y genéticos.Resultados: En 26 centros se incluyó a 181 pacientes, el 65,2% varones, con una mediana de edad al diagnóstico de 62 años. Las mutaciones más frecuentes fueron Val50Met (67,7%) y Val142Ile (12,4%). El principal motivo de consulta fue extracardiaco (69%), principalmente neurológico. La media de la fracción aminoterminal del propéptido natriurético cerebral (NT-proBNP) fue 2.145±3.586 pg/ml. Lo más característico del electrocardiograma fueron el patrón de seudoinfarto (25,9%) y el bloqueo auriculoventricular (25,3%). El grosor ventricular medio fue 15,4±4,1mm. El strain longitudinal estaba reducido en segmentos basales en el 29,4%. Se observó realce tardío subendocárdico difuso en el 58,8%. En la gammagrafía había captación de grados 2-3 en un 75%. En el seguimiento, el 24,9% ingresó por insuficiencia cardiaca, el 34,3% precisó marcapasos y el 31,6%, trasplante hepático. El 32,5% falleció, principalmente por insuficiencia cardiaca (28,8%). Las mutaciones diferentes de Val50Met se asociaron en general con un peor pronóstico. Conclusiones: La ATTRv cardiaca en España tiene un espectro genético y de afección heterogéneo. El pronóstico es malo principalmente por las complicaciones cardiacas, por lo que son esenciales un diagnóstico y un tratamiento precoces (AU)

Introduction and objectives: Hereditary transthyretin amyloidosis (hATTR) is a disease caused by mutations in the transthyretin gene that frequently shows cardiac involvement due to amyloid deposition in the myocardium. Our objective was to identify cardiac involvement in a Spanish cohort. Methods: Retrospective multicenter study of patients diagnosed with hATTR with cardiac involvement from Spanish centers. We collected demographic, clinical, and genetic data. Result: A total of 181 patients from 26 centers were included (65.2% men, with a median age at diagnosis of 62 years). The most frequent mutations were Val50Met (67.7%) and Val142Ile (12.4%). The main reason for consultation was extracardiac symptoms (69%), mainly neurological. The mean N-terminal pro-B-type natriuretic peptide level was 2145±3586 pg/mL. The most characteristic electrocardiogram findings were a pseudoinfarct pattern (25.9%) and atrioventricular block (25.3%). Mean ventricular thickness was 15.4±4.1mm. Longitudinal strain was reduced in basal segments by 29.4%. Late diffuse subendocardial enhancement was observed in 58.8%. Perugini grade 2 or 3 uptake was observed in 75% of scintigraphy scans. During follow-up, 24.9% of the patients were admitted for heart failure, 34.3% required a pacemaker, and 31.6% required a liver transplant. One third (32.5%) died during follow-up, mainly due to heart failure (28.8%). The presence of non-Val50Met mutations was associated with a worse prognosis.Conclusions: HATTR cardiac amyloidosis in Spain shows heterogeneous genetic and clinical involvement. The prognosis is poor, mainly due to cardiac complications. Consequently early diagnosis and treatment are vital (AU)

Lysozyme amyloidosis-a report on a large German cohort and the characterisation of a novel amyloidogenic lysozyme gene variant.

Lysozyme-derived (ALys) amyloidosis is a rare type of hereditary amyloidosis. Nine amyloidogenic variants and ∼30 affected families have been described worldwide. The most common manifestations are renal dysfunction, gastrointestinal tract symptoms, and sicca syndrome. We report on the clinical course of ten patients from six families representing one of the largest cohorts published so far. Seven patients carried the W64R variant showing the whole spectrum of ALys-associated symptoms. Two patients-a mother-son pair-carried a novel lysozyme variant, which was associated with nephropathy and peripheral polyneuropathy. In accordance with previous findings, the phenotype resembled within these families but did not correlate with the genotype. To gain insights into the effect of the variants at the molecular level, we analysed the structure of lysozyme and performed comparative computational predictions on aggregation propensity and conformational stability. Our study supports that decreased conformational stability is a key factor for lysozyme variants to be prone to aggregation. In summary, ALys amyloidosis is a very rare, but still heterogeneous disease that can manifest at an early age. Our newly identified lysozyme variant is associated with nephropathy and peripheral polyneuropathy. Further research is needed to understand its pathogenesis and to enable the development of new treatments.

The experience of hereditary apolipoprotein A-I amyloidosis at the UK National Amyloidosis Centre.

INTRODUCTION: Hereditary apolipoprotein A-I (AApoAI) amyloidosis is a rare heterogeneous disease with variable age of onset and organ involvement. There are few series detailing the natural history and outcomes of solid organ transplantation across a range of causative APOA1 gene mutations. METHODS: We identified all patients with AApoAI amyloidosis who presented to the National Amyloidosis Centre (NAC) between 1986 and 2019. RESULTS: In total, 57 patients with 14 different APOA1 mutations were identified including 18 patients who underwent renal transplantation (5 combined liver-kidney (LKT) and 2 combined heart-kidney (HKT) transplants). Median age of presentation was 43 years and median time from presentation to referral was 3 (0-31 years). Involvement of the kidneys, liver and heart by amyloid was detected in 81%, 67% and 28% of patients, respectively. Renal amyloidosis was universal in association with the most commonly identified variant (Gly26Arg, n = 28). Across all variants, patients with renal amyloidosis had a median creatinine of 159 µmol/L and median urinary protein of 0.3 g/24 h at the time of diagnosis of AApoAI amyloidosis and median time from diagnosis to end-stage renal disease was 15.0 (95% CI: 10.0-20.0) years. Post-renal transplantation, median allograft survival was 22.0 (13.0-31.0) years. There was one early death following transplantation (infection-related at 2 months post-renal transplant) and no episodes of early rejection leading to graft failure. Liver transplantation led to regression of amyloid in all four cases in whom serial 123I-SAP scintigraphy was performed. CONCLUSIONS: AApoAI amyloidosis is a slowly progressive disease that is challenging to diagnose. The outcomes of transplantation are encouraging and graft survival is excellent.

A novel substitution of proline (P32L) destabilises ß2-microglobulin inducing hereditary systemic amyloidosis.

BACKGROUND: ß2-microglobulin amyloidosis was first described in the 1980s as a protein deposition disease associated with long-term haemodialysis. More recently, two inherited forms resulting from separate point mutations in the ß2-microglobulin gene have been identified. In this report, we detail a novel ß2M variant, P32L, caused by a unique dinucleotide mutation that is linked to systemic hereditary ß2-microglobulin amyloidosis. METHODS: Three family members from a Portuguese kinship featured cardiomyopathy, requiring organ transplantation in one case, along with soft tissue involvement; other involvements included gastrointestinal, neuropathic and sicca syndrome. In vitro studies with recombinant P32L, P32G, D76N and wild-type ß2-microglobulin were undertaken to compare the biophysical properties of the proteins. RESULTS: The P32L variant was caused by the unique heterozygous dinucleotide mutation c.154_155delinsTT. Amyloid disease featured lowered serum ß2-microglobulin levels with near equal amounts of circulating P32L and wild-type proteins; amyloid deposits were composed exclusively of P32L variant protein. In vitro studies of P32L demonstrated thermodynamic and chemical instability and enhanced susceptibility to proteolysis with rapid formation of pre-fibrillar oligomeric structures by N- and C-terminally truncated species under physiological conditions. CONCLUSIONS: This work provides both clinical and experimental evidence supporting the critical role of P32 residue replacement in ß2M amyloid fibrillogenesis.

Combined Heart-Liver and Domino Liver Transplantation in Familial Amyloidosis.

BACKGROUND: Combined heart-liver transplantation (CHLT) is the only curative option for patients with concomitant pathology affecting the heart and liver. In some cases, the native livers of familial amyloidosis (FA) patients may be suitable for domino transplantation into other recipients. METHODS: Retrospective analysis (2013 to 2019) of all CHLT at our center was performed. Continuous data were presented as mean with standard deviation and discrete variables as percentages. RESULTS: Familial amyloidosis was the indication for CHLT in 5 out of 6 patients. The mean recipient age was 55 ± 5.62 years. Two patients were bridged with total artificial heart. The mean model for end-stage liver disease score at transplant was 17.17 ± 3.7. Two explanted livers were used for transplantation in a domino fashion. The median intensive care and hospital stays were 5.5 and 19 days, respectively. Complications included renal failure (1), groin abscess (1), pulmonary embolism (1), and cardiac rejection (1). Patient and graft survival for both organs was 100% at a median follow-up of 59 (range 20-76) months. DISCUSSION: Combined heart-liver transplantation for FA achieves excellent outcomes. The possible use of livers explanted from patients with FA for domino liver transplantation can contribute to the liver donor pool.

Amyloidosis cutis dyschromica cases caused by GPNMB mutations with different inheritance patterns.

BACKGROUND: Amyloidosis cutis dyschromica (ACD) is a rare form of primary cutaneous amyloidosis featured by reticulate dotted hypo- and hyperpigmentation. Recently, loss-of-function mutations in GPNMB, encoding glycoprotein (transmembrane) nonmetastatic melanoma protein B, were found in autosomal-recessive or semi-dominant ACD. OBJECTIVE: This study aims to detect the genetic defect underlying ACD in nine separate cases and to investigate the functional consequences of the mutants. METHODS: Nine ACD cases were collected including eight with autosomal-recessive pattern and one with autosomal-dominant pattern. Whole-exome sequencing or Sanger sequencing of the GPNMB gene was performed to detect the pathogenic mutations. Haplotype analysis was employed to determine the origin of mutation c.565C > T using adjacent highly polymorphic SNPs. Immunoblotting and subcellular localization assessments were performed to evaluate the expression of the mutants using HEK293 cells transfected with the GPNMB constructs. RESULTS: We detected four recurrent mutations (c.393 T > G, p.Y131*; c.565C > T, p.R189*; c.1056delT, p.P353Lfs*20; c.1238 G > C, p.C413S) and two novel mutations (c.935delA, p.N312Tfs*4; c.969 T > A, p.C323*) in GPNMB. Mutation c.565C > T found in six separate ACD cases shared a common haplotype. The two novel mutations caused a decreased abundance of truncated proteins. The c.1238 G > C mutation, which was detected in the autosomal-dominant case, caused abnormal reticular subcellular localization of the protein. A major percentage of wildtype changed its expression pattern when co-expressed with this mutant. CONCLUSIONS: Our findings proved that the recurrent mutation c.565C > T originated from a founder effect. The autosomal-dominant ACD associated mutation p.C413S played its pathogenic role through a dominant-negative effect on wild-type GPNMB. This study expands the genotype and inherited modes of ACD and improves our understanding of the pathogenesis of this disorder.

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.

Two missense mutations in GPNMB cause autosomal recessive amyloidosis cutis dyschromica in the consanguineous pakistani families.

BACKGROUND: Amyloidosis cutis dyschromica (ACD) is a rare variant of cutaneous amyloidosis. This disorder often clusters in families, and it has been suggested that genetic factors might be involved in its development. OBJECTIVE: To identify the genetic causes of ACD, we recruited a consanguineous Pakistani family with multiple cases of ACD that display a recessive mode of inheritance. METHODS: We performed whole-exome sequencing of samples from 7 members of this family, followed by bioinformatic and in silico analyses to identify the causative variant. For the replication study, we recruited a British family with Pakistani ancestry, and sequenced all exons of glycoprotein non-metastatic melanoma protein b (GPNMB) to identify mutations. We also investigated effects of the mutations on the stability of the GPNMB protein using the I-TASSER three-dimensional modeling tool. RESULTS: We found a novel homozygous mutation, p.Gly363Val (c.1088 G>T), in GPNMB in all affected cases. In a replication study, another homozygous missense mutation in GPNMB, pIle174Met (c.522 C>G), was carried by the affected son. The two mutations were not observed in our in-house data set comprising 217 healthy Pakistani individuals or in The Genome Aggregation Database. Our structural modeling of GPNMB suggested that p.Gly363Val enhanced its stability, whereas p.Ile174Met caused instability. CONCLUSIONS: This study reports two novel missense mutations in two Pakistani families that cause ACD. The mutations appear to influence GPNMB stability, as revealed by protein modeling.

[Amyloidosis cutis dyschromica due to homozygous variants of the GPNMB gene in a Chinese pedigree].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with amyloidosis cutis dyschromica. METHODS: High-throughput sequencing was carried out for the proband. Bioinformatic analysis was used to identify the pathogenic variants. The result was verified by Sanger sequencing. RESULTS: A homozygous nonsense variant c.565C>T (p.Arg189X) of the GPNMB gene was identified in the proband, his elder brother and younger sister, which resulted a truncated protein with loss of function. The father of the proband was a heterozygous carrier for the variant. The genotype of his mother was unknown since she had passed away. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.565C>T variant was predicted to be likely pathogenic (PS3+ PM2+ PP1+PP3). CONCLUSION: The novel homozygous GPNMB variant probably underlay the amyloidosis cutis dyschromica in this pedigree. Above finding has expanded the spectrum of GPNMB gene variants.