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Hyperlysinemia is a rare autosomal recessive deficiency of 2-aminoadipic semialdehyde synthase (AASS) affecting the initial step in lysine degradation. It is thought to be a benign biochemical abnormality, but reports on cases remain scarce. The description of additional cases, in particular, those identified without ascertainment bias, may help counseling of new cases in the future. It may also help to establish the risks associated with pharmacological inhibition of AASS, a potential therapeutic strategy that is under investigation for other inborn errors of lysine degradation. We describe the identification of a hyperlysinemia case identified in the Provincial Neonatal Urine Screening Program in Sherbrooke, Quebec. This case presented with a profile of cystinuria but with a very high increase in urinary lysine. A diagnosis of hyperlysinemia was confirmed through biochemical testing and the identification of biallelic variants in AASS. The p.R146W and p.T371I variants are novel and affect the folding of the lysine-2-oxoglutarate domain of AASS. The 11-month-old boy is currently doing well without any therapeutic interventions. The identification of this case through newborn urine screening further establishes that hyperlysinemia is a biochemical abnormality with limited clinical consequences and may not require any intervention.
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Background: A biomarker profile was evaluated longitudinally in patients with Fabry disease switched from enzyme-replacement therapy (ERT) to migalastat. Methods: 16 Gb3 isoforms and eight lyso-Gb3 analogues were analyzed in plasma and urine by LC-MS/MS at baseline and at three different time points in naive participants and participants switching from either agalsidase α or ß to migalastat. Results: 29 adult participants were recruited internationally (seven centers). The Mainz Severity Score Index and mean biomarker levels remained stable (p ≥ 0.05) over a minimum of 12 months compared with baseline following the treatment switch. Conclusion: In this cohort of patients with Fabry disease with amenable mutations, in the short term, a switch from ERT to migalastat did not have a marked effect on the average biomarker profile.
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Doença de Fabry , Adulto , Humanos , Doença de Fabry/tratamento farmacológico , Doença de Fabry/genética , Cromatografia Líquida , Espectrometria de Massas em Tandem , 1-Desoxinojirimicina/uso terapêutico , BiomarcadoresRESUMO
OBJECTIVE: To present phenotypic characteristics and biomarkers of a family with the rare mutation Thr410Ala of the α-galactosidase A gene (T410A/GLA) causing Fabry disease (FD). METHODS AND RESULTS: In a woman in her 60s with hypertrophic cardiomyopathy, T410A/GLA was found in screening for variants in 59 cardiomyopathy-related genes. Her son in his 40s, two granddaughters and two great grandsons carried T410A/GLA. The son had a history of hypertension and paroxysmal AF but no microalbuminuria or classic symptoms or signs of FD. Baseline α-galactosidase A enzyme (α-Gal A) activity varied from 0% to 26.5%. Cardiac MRI showed mild Fabry cardiomyopathy (FC). During 11 years of enzyme replacement therapy (ERT), FC progressed and he suffered sudden cardiac death in his 50s. The great grandsons with T410A/GLA had no active α-Gal A, high lyso-Gb3 levels and normal cardiac imaging. They suffered from neuropathic pain and gastrointestinal symptoms and were started with ERT at the age under 10. Granddaughters with T410A/GLA had α-Gal A activities of 8-18 and 10% of normal. The older granddaughter in her 30s was diagnosed with incipient FC. Plasma lyso-Gb3 analogues were elevated, markedly in the elder male with FC and moderately in the elder granddaughter. In young males with classic phenotype, plasma lyso-Gb3 analogues were only slightly elevated. CONCLUSIONS: The T410A/GLA mutation caused late-onset FD with progressive cardiomyopathy in elder male, and classic FD in young males of the same family. Varying levels of α-Gal A and lyso-Gb3 analogues reflected variable phenotype of FD in the family.
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Cardiomiopatia Hipertrófica , Doença de Fabry , Feminino , Masculino , Humanos , Doença de Fabry/complicações , Doença de Fabry/diagnóstico , Doença de Fabry/genética , alfa-Galactosidase/genética , Mutação , FenótipoRESUMO
The safety and efficacy of lentivirus-mediated gene therapy was recently demonstrated in five male patients with Fabry disease-a rare X-linked lysosomal storage disorder caused by GLA gene mutations that result in multiple end-organ complications. To evaluate the risks of clonal dominance and leukemogenesis, which have been reported in multiple gene therapy trials, we conducted a comprehensive DNA insertion site analysis of peripheral blood samples from the five patients in our gene therapy trial. We found that patients had a polyclonal integration site spectrum and did not find evidence of a dominant clone in any patient. Although we identified vector integrations near proto-oncogenes, these had low percentages of contributions to the overall pool of integrations and did not persist over time. Overall, we show that our trial of lentivirus-mediated gene therapy for Fabry disease did not lead to hematopoietic clonal dominance and likely did not elevate the risk of leukemogenic transformation.
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Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired α-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer®B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3-52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD.
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Doença de Fabry , Glicolipídeos , Humanos , Glicolipídeos/química , Espectrometria de Massas em Tandem/métodos , Esfingolipídeos , Doença de Fabry/diagnóstico , alfa-Galactosidase/metabolismo , BiomarcadoresRESUMO
The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Glicoesfingolipídeos/biossíntese , Humanos , Células Tumorais CultivadasRESUMO
Enzyme and chaperone therapies are used to treat Fabry disease. Such treatments are expensive and require intrusive biweekly infusions; they are also not particularly efficacious. In this pilot, single-arm study (NCT02800070), five adult males with Type 1 (classical) phenotype Fabry disease were infused with autologous lentivirus-transduced, CD34+-selected, hematopoietic stem/progenitor cells engineered to express alpha-galactosidase A (α-gal A). Safety and toxicity are the primary endpoints. The non-myeloablative preparative regimen consisted of intravenous melphalan. No serious adverse events (AEs) are attributable to the investigational product. All patients produced α-gal A to near normal levels within one week. Vector is detected in peripheral blood and bone marrow cells, plasma and leukocytes demonstrate α-gal A activity within or above the reference range, and reductions in plasma and urine globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are seen. While the study and evaluations are still ongoing, the first patient is nearly three years post-infusion. Three patients have elected to discontinue enzyme therapy.
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Doença de Fabry/enzimologia , Doença de Fabry/terapia , Terapia Genética/métodos , Lentivirus/genética , alfa-Galactosidase/genética , alfa-Galactosidase/uso terapêutico , Adulto , Antígenos CD34 , Células da Medula Óssea , Doença de Fabry/genética , Vetores Genéticos , Células-Tronco Hematopoéticas , Humanos , Leucócitos , Masculino , Pessoa de Meia-Idade , Triexosilceramidas/sangue , Triexosilceramidas/urinaRESUMO
PURPOSE: To assess the utility of globotriaosylsphingosine (lyso-Gb3) for clinical monitoring of treatment response in patients with Fabry disease receiving migalastat. METHODS: A post hoc analysis evaluated data from 97 treatment-naive and enzyme replacement therapy (ERT)-experienced patients with migalastat-amenable GLA variants from FACETS (NCT00925301) and ATTRACT (NCT01218659) and subsequent open-label extension studies. The relationship between plasma lyso-Gb3 and measures of Fabry disease progression (left ventricular mass index [LVMi], estimated glomerular filtration rate [eGFR], and pain) and the relationship between lyso-Gb3 and incidence of Fabry-associated clinical events (FACEs) were assessed in both groups. The relationship between changes in lyso-Gb3 and kidney interstitial capillary (KIC) globotriaosylceramide (Gb3) inclusions was assessed in treatment-naive patients. RESULTS: No significant correlations were identified between changes in lyso-Gb3 and changes in LVMi, eGFR, or pain. Neither baseline lyso-Gb3 levels nor the rate of change in lyso-Gb3 levels during treatment predicted FACE occurrences in all patients or those receiving migalastat for ≥24 months. Changes in lyso-Gb3 correlated with changes in KIC Gb3 inclusions in treatment-naive patients. CONCLUSIONS: Although used as a pharmacodynamic biomarker in research and clinical studies, plasma lyso-Gb3 may not be a suitable biomarker for monitoring treatment response in migalastat-treated patients.
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Doença de Fabry , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapêutico , Terapia de Reposição de Enzimas , Doença de Fabry/diagnóstico , Doença de Fabry/tratamento farmacológico , Doença de Fabry/genética , Humanos , alfa-Galactosidase/genéticaRESUMO
INTRODUCTION: Recent studies showed the usefulness of globotriaosylsphingosine (lyso-Gb3) and related analogues, deacylated forms of globotriaosylceramide (Gb3), for high-risk screening, treatment monitoring and follow-up for patients with Fabry disease. METHODS: We evaluated Gb3, lyso-Gb3 and analogues using tandem mass spectrometry in 57 women with Fabry disease followed during a period of 15.4 years. Twenty-one women were never treated and 36 received treatment (agalsidase-beta, n=30; agalsidase-alfa, n=5; or migalastat, n=1). Lyso-Gb3 and analogues at m/z (-28), (-2), (+16), (+34) and (+50) were analysed in plasma and urine. Total Gb3 and lyso-Gb3 analogues at m/z (-12) and (+14) were evaluated in urine while the analogue at m/z (+18) was evaluated in plasma. RESULTS: A strong correlation between plasma and urine lyso-Gb3 and analogue levels was revealed. Plasma and urine lyso-Gb3 and analogue levels were not statistically different between patients carrying missense (n=49), nonsense (n=6) or deletion mutations (n=2). Never treated patients had lower plasma lyso-Gb3 and analogues at m/z (-28), (-2), (+16), (+34) and the seven urinary lyso-Gb3 analogues compared with pretreatment levels of the treated patients. A significant reduction of plasma lyso-Gb3 and five analogues, as well as urine Gb3 and six lyso-Gb3 analogues, but not lyso-Gb3 and lyso-Gb3 at m/z (+50), was observed post-treatment with agalsidase-beta. The same tendency was observed with agalsidase-alfa. CONCLUSION: Women with Fabry disease who started treatment based on clinical manifestations had higher lyso-Gb3 and analogue biomarker levels than never treated women. This indicates that a biomarker cut-off could potentially be a decision tool for treatment initiation in women with Fabry disease.
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Doença de Fabry/sangue , Doença de Fabry/diagnóstico , Glicolipídeos/sangue , Glicolipídeos/urina , Esfingolipídeos/sangue , Esfingolipídeos/urina , Alelos , Substituição de Aminoácidos , Biomarcadores , Estudos de Coortes , Dinamarca/epidemiologia , Gerenciamento Clínico , Terapia de Reposição de Enzimas , Doença de Fabry/genética , Doença de Fabry/terapia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Heterozigoto , Humanos , Resultado do Tratamento , alfa-Galactosidase/genéticaRESUMO
Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb3 isoforms (various fatty acids) and lyso-Gb3 analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb3 and lyso-Gb3 levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb3 and particular lyso-Gb3 analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb3 and lyso-Gb3 isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb3 massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb3 accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb3 or lyso-Gb3 may play major roles in the pathogenesis of Fabry disease, and that Gb3 and lyso-Gb3 are not responsible for the pathology in all tissues or cell types.
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Modelos Animais de Doenças , Doença de Fabry/metabolismo , Glicoesfingolipídeos/metabolismo , Animais , Doença de Fabry/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Índice de Gravidade de DoençaRESUMO
Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the α-galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (-C2H4, -C2H4+O, -H2, -H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-naïve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.
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Doença de Fabry/tratamento farmacológico , Glicolipídeos/urina , Esfingolipídeos/urina , Triexosilceramidas/urina , alfa-Galactosidase/administração & dosagem , Adulto , Biomarcadores/urina , Estudos de Casos e Controles , Ritmo Circadiano , Esquema de Medicação , Cronofarmacoterapia , Terapia de Reposição de Enzimas , Doença de Fabry/urina , Feminino , Humanos , Infusões Intravenosas , Masculino , Resultado do Tratamento , alfa-Galactosidase/uso terapêuticoAssuntos
Doença de Fabry/complicações , Gastroenteropatias/tratamento farmacológico , alfa-Galactosidase/administração & dosagem , Administração Oral , Adulto , Estudos de Coortes , Suplementos Nutricionais , Doença de Fabry/tratamento farmacológico , Feminino , Gastroenteropatias/etiologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder with a highly heterogeneous clinical presentation. This complex disease is caused by a deficient activity of the enzyme α-galactosidase A, which is involved in the catabolism of glycosphingolipids. The prevalence of Fabry disease is underestimated, due to the presence of atypical variants. High-risk screening protocols are particularly relevant for this disease due to the availability of treatments, such as enzyme replacement and chaperone therapies. As kidney manifestations are present in the majority of male and many female patients with Fabry disease, a high-risk screening protocol was performed for patients with chronic kidney disease of unknown etiology. METHODS: Recruitment of 397 participants took place in four centers across Canada from 2011 to 2017. Globotriaosylceramide (Gb3) was analyzed in dried urine spots by liquid chromatography/tandem mass spectrometry followed by globotriaosylsphingosine (lyso-Gb3) on the repeat analysis. RESULTS: The collection and shipment of urine specimens on filter paper resulted in easier handling/shipment and significant cost-saving. No Fabry patients were detected in this study. CONCLUSIONS: Increased concentrations of urinary Gb3 were observed in 13.6% of patients with chronic kidney disease suggesting that chronic kidney disease or other comorbidities might be associated with increased urinary Gb3 concentrations.
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Doença de Fabry/diagnóstico , Ensaios de Triagem em Larga Escala , Insuficiência Renal Crônica/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Canadá , Estudos de Coortes , Doença de Fabry/urina , Feminino , Glicolipídeos/urina , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/urina , Fatores de Risco , Esfingolipídeos/urina , Triexosilceramidas/urina , Adulto JovemRESUMO
BACKGROUND: Fabry disease (FD) is a genetic disorder caused by defective α-galactosidase-A enzyme due to mutations in the GLA gene. A reliable diagnosis in classical FD males can be made by measuring the enzyme activity while diagnosing classical FD females and non-classical FD patients requires mutation analysis. Plasma globotriaosylsphingosine (Lyso-Gb3) has progressively gained more importance as a diagnostic biomarker for FD in recent years. Having another biomarker to complement plasma Lyso-Gb3 will increase the diagnostic accuracy in the era of mass screening, and also precision medicine. This study aims to highlight the clinical utility of the total concentration of urinary Lyso-Gb3 plus its analogues in diagnosing FD. METHOD: Random urine samples collected from 42 FD patients and 48 healthy individuals. Lyso-Gb3 and its analogues were enriched by solid phase extraction and analysed by liquid chromatography tandem mass spectrometry. RESULTS: The total concentration of Lyso-Gb3 plus its analogues in classical FD male and female patients were 1124.4⯱â¯181.2 and 308.6⯱â¯78.6â¯pmol/mmol creat., respectively. The levels in non-classical FD male and female patients were 229.2⯱â¯169.4 and 314.4⯱â¯156.4â¯pmol/mmol creat., respectively. Urinary Lyso-Gb3 and its analogues were virtually undetectable in healthy volunteers making it 100% specific for FD diagnosis. For FD male and female patients, total concentration of urinary Lyso-Gb3 plus its analogue levels ≥0.25â¯pmol/mmol creat. yielded a diagnostic sensitivity and specificity of 100% (AUCâ¯=â¯1, pâ¯<â¯0.00001). CONCLUSIONS: Our study findings support that the total concentration of Lyso-Gb3 plus its analogues in urine is specific to FD and may provide extra diagnostic utility for both classical and non-classical FD patients.
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Doença de Fabry/diagnóstico , Doença de Fabry/urina , Esfingolipídeos/química , Esfingolipídeos/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Mucopolysaccharidoses (MPSs) are lysosomal storage disorders caused by deficiencies of enzymes involved in the catabolism of glycosaminoglycans (GAGs). Various treatments such as enzyme replacement therapy and/or hematopoietic stem cell transplant are available for MPSs. Early initiation of treatment improves the outcome and delays the onset of symptoms, highlighting the need for newborn screening for MPSs. The main objective of this project was to devise and validate a multiplex urine filter paper method for GAG analysis using a tandem mass spectrometry (MS/MS) approach to screen newborns for MPSs. Eluted urine samples from 21-day-old newborns were evaporated and a methanolysis reaction was performed. Samples were resuspended and analyzed using a UPLC-MS/MS system. A one-minute chromatographic method allowed the absolute quantification of heparan sulfate (HS), dermatan sulfate (DS), and creatinine. Method validation revealed high precision (< 9% relative standard deviation) and accuracy (< 7% bias) for all analytes. The reference values normalized to creatinine obtained by the analysis of five hundred 21-day-old newborn urine samples were 34.6 +/-6.2 mg/mmol of creatinine and 17.3 +/-3.9 mg/mmol of creatinine for HS and DS, respectively. We present a rapid and efficient method for populational newborn urine screening using MS/MS, which could also be applied to high-risk screening.
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BACKGROUND: Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD. METHODS: Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy. RESULTS: Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells. CONCLUSION: We conclude that repeated AGAL activity measurements in patients' white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.
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Doença de Fabry/genética , alfa-Galactosidase/genética , 1-Desoxinojirimicina/administração & dosagem , 1-Desoxinojirimicina/análogos & derivados , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia de Reposição de Enzimas/métodos , Doença de Fabry/metabolismo , Doença de Fabry/terapia , Edição de Genes , Células HEK293 , Humanos , Chaperonas Moleculares/administração & dosagem , Medicina de Precisão/métodos , Triexosilceramidas/metabolismo , alfa-Galactosidase/metabolismoRESUMO
BACKGROUND: Podocytes are highly differentiated visceral cells, and several related specific proteins, such as podocalyxin and podocin are potential tools for the evaluation of podocyturia. However, precise quantitation of podocyturia-related proteins is complex and often unreliable. METHOD: A reversed-phase ultra-performance liquid chromatography coupled to tandem mass spectrometry method was developed and validated to quantify podocalyxin and podocin levels in urine supernatant by using specific cleavable peptides and standards. Urine samples from women with normotensive or hypertensive pregnancies, gestational diabetes and preeclampsia, as well as treated and untreated Fabry patients, and gender-matched controls were investigated. RESULTS: The multiplex analysis shows that podocalyxin levels were higher than podocin levels in patients, the former being particularly higher in pregnant women. Women with preeclampsia had abnormal urine levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were also observed in Fabry males, while both proteins were increased in untreated Fabry females. Correlations were established between podocalyxin and podocin levels and clinical parameters associated with Fabry disease and preeclampsia. CONCLUSIONS: This methodology makes possible the precise, simultaneous and reliable analysis of podocalyxin and podocin levels, and offers a valuable tool for the evaluation of podocyturia.
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Doença de Fabry/patologia , Peptídeos e Proteínas de Sinalização Intracelular/urina , Proteínas de Membrana/urina , Podócitos/patologia , Pré-Eclâmpsia/patologia , Sialoglicoproteínas/urina , Espectrometria de Massas em Tandem , Urinálise/métodos , Calibragem , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Doença de Fabry/urina , Feminino , Humanos , Pré-Eclâmpsia/urina , Gravidez , Reprodutibilidade dos TestesRESUMO
Background Because systemic inflammation and endothelial dysfunction lead to heart failure with preserved ejection fraction, we characterized plasma levels of inflammatory and cardiac remodeling biomarkers in patients with Fabry disease ( FD ). Methods and Results Plasma biomarkers were studied in multicenter cohorts of patients with FD (n=68) and healthy controls (n=40). Plasma levels of the following markers of inflammation and cardiac remodeling were determined: tumor necrosis factor ( TNF ), TNF receptor 1 ( TNFR 1) and 2 ( TNFR 2), interleukin-6, matrix metalloprotease-2 ( MMP -2), MMP -8, MMP -9, galectin-1, galectin-3, B-type natriuretic peptide ( BNP ), midregional pro-atrial natriuretic peptide ( MR -pro ANP ), and globotriaosylsphingosine. Clinical profile, cardiac magnetic resonance imaging, and echocardiogram were reviewed and correlated with biomarkers. Patients with FD had elevated plasma levels of BNP , MR -pro ANP , MMP -2, MMP -9, TNF , TNFR 1, TNFR 2, interleukin-6, galectin-1, globotriaosylsphingosine, and analogues. Plasma TNFR 2, TNF , interleukin-6, MMP -2, and globotriaosylsphingosine were elevated in FD patients with left ventricular hypertrophy, whereas diastolic dysfunction correlated with higher BNP , MR -pro ANP , and MMP -2 levels. Patients with late gadolinium enhancement on cardiac magnetic resonance imaging had greater levels of BNP , MR -pro ANP , TNFR 1, TNFR 2, and MMP -2. Plasma BNP , MR -pro ANP , MMP -2, MMP -8, TNF , TNFR 1, TNFR 2, galectin-1, and galectin-3 were elevated in patients with renal dysfunction. Patients undergoing enzyme replacement therapy who have more severe disease had higher MMP -2, TNF , TNFR 1, TNFR 2, and globotriaosylsphingosine analogue levels. Conclusions Inflammatory and cardiac remodeling biomarkers are elevated in FD patients and correlate with disease progression. These features are consistent with a phenotype dominated by heart failure with preserved ejection fraction and suggest a key pathogenic role of systemic inflammation in FD .
Assuntos
Doença de Fabry/sangue , Doença de Fabry/complicações , Insuficiência Cardíaca/etiologia , Adulto , Remodelamento Atrial , Biomarcadores/sangue , Doença de Fabry/fisiopatologia , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Inflamação/sangue , Inflamação/complicações , Masculino , Pessoa de Meia-Idade , Volume SistólicoRESUMO
Fabry disease is an X-linked lysosomal storage disorder with marked variability in the phenotype and genotype. Glycosphingolipids such as globotriaosylceramide (Gb3) isoforms/analogs, globotriaosylsphingosine (lyso-Gb3) and analogs, and galabiosylceramide (Ga2) isoforms/analogs may accumulate in biological fluids and different organs. The aims of this study were to: 1) develop/validate a novel UHPLC-MS/MS method for relative quantitation of Gb3 in leukocytes (unfractionated white blood cells), B lymphocytes and monocytes; 2) evaluate these biomarkers in a cohort of Fabry patients and healthy controls; and 3) assess correlations between these biomarkers, treatment and genotype. Whole blood, plasma and urine samples from 21 Fabry patients and 20 healthy controls were analyzed. Samples were purified by liquid-liquid extraction and analyzed by UHPLC-MS/MS in positive electrospray ionization. Methylated Gb3 isoforms were detected, showing that a methylation process occurs at the cellular level. Our results show that there were no significant differences in the distribution of the different Gb3 isoforms/analogs in blood cells between Fabry patients and healthy controls. In leukocyte, Gb3[(d18:1)(C14:0)], Gb3[(d18:1)(C16:0)], Gb3 [(d18:1)(C16:0)]Me, Gb3 [(d18:1)(C16:1)], Gb3 [(d18:1)(C18:0)], Gb3 [(d18:1)(C18:1)], Gb3 [(d18:1)(C20:1)], Gb3 [(d18:1)(C24:2)], Gb3 [(d18:1)(C26:1)] and total Gb3 allowed good discrimination between male Fabry patients and male controls, patients having higher biomarker levels than controls. Regarding B lymphocytes and monocytes, the same tendency was observed without reaching statistical significance. A positive concordance between mutation types and biomarker levels in white blood cells was established. Our results might provide a deeper mechanistic comprehension of the underlying biochemical processes of Gb3 biomarkers in white blood cells of Fabry patients.
Assuntos
Doença de Fabry/diagnóstico , Leucócitos/química , Triexosilceramidas/análise , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Fabry disease (FD) is a multi-systemic X-linked lysosomal disorder caused by the deficient activity of α-galactosidase-A enzyme, which leads to accumulation of glycosphingolipids in various body tissues. The N215S mutation is a known variant of FD, with a late onset cardiac phenotype. Consensus guidelines acknowledged the use of globotriaosylsphingosine (Lyso-Gb3) as a diagnostic marker for classical FD but its utility for cardiac variant FD is not clear. We aim to characterize the clinical features and evaluate the diagnostic accuracy of plasma and urinary Lyso-Gb3 levels in N215S cardiac variant FD patients. Thirty-four FD patients with the late-onset N215S cardiac variant mutation were enrolled along with 62 classical FD patients and 109 healthy controls. Plasma and urinary Lyso-Gb3 and its analogues were analyzed by LC-MS/MS. Both FD males and females with N215S mutation showed Lyso-Gb3 levels of (mean ± SEM) 9.7 ± 1.0 and 5.4 ± 0.8 nM, respectively. These levels were significantly higher than healthy control and lower than classical FD patients (p < 0.0001). Plasma Lyso-Gb3 levels equal to or higher than 2.7 nM yielded a diagnostic sensitivity and specificity of 100% (AUC = 1, p < 0.0001). Cardiac involvement was frequent with 16/34 (47%) developing left ventricular hypertrophy. Three patients who underwent renal biopsy had the characteristic sphingolipid deposition in the podocytes while 6/19 (32%) had evidence of white matter changes or infarct on brain MRI. Taken together, cardiac variant N215S mutation is rather an attenuated form of classical FD. Plasma Lyso-Gb3 is a diagnostic hallmark to differentiate N215S variant phenotype from subjects with no FD.