scholarly journals Assessment of Gene Variant Amenability for Pharmacological Chaperone Therapy with 1-Deoxygalactonojirimycin in Fabry Disease

2020 ◽  
Vol 21 (3) ◽  
pp. 956 ◽  
Author(s):  
Jan Lukas ◽  
Chiara Cimmaruta ◽  
Ludovica Liguori ◽  
Supansa Pantoom ◽  
Katharina Iwanov ◽  
...  
Keyword(s):  
Cell Culture ◽  
Enzyme Activity ◽  
Fabry Disease ◽  
Response Threshold ◽  
Clinical Indication ◽  
Gene Variants ◽  
Absolute Increase ◽  
Pharmacological Chaperone ◽  
Gla Gene ◽  
The Impact

Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal α-galactosidase A (α-Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the α-galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of ≥3% of wild type enzyme activity and a relative increase in enzyme activity of ≥1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own α-galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.

p.G360R Is a Pathogenic GLA Gene Mutation Responsible for a Classic Phenotype of Fabry Disease

Cardiology ◽  
2019 ◽  
Vol 144 (3-4) ◽  
pp. 125-130 ◽  
Author(s):  
Daniela Marisa Carvalho Silva ◽  
Nuno Marques ◽  
Olga Azevedo ◽  
Gabriel Miltenberger-Miltenyi ◽  
Dina Bento ◽  
...  
Keyword(s):  
Left Ventricular Hypertrophy ◽  
Fabry Disease ◽  
Ventricular Hypertrophy ◽  
Left Ventricular ◽  
Gene Variants ◽  
Fabry Patient ◽  
Enzyme Replacement ◽  
Disease Awareness ◽  
Classic Phenotype ◽  
Gla Gene

The authors report the case of a classic phenotype of Fabry disease in a 60-year-old male patient presenting with left ventricular hypertrophy and stroke. Genetic analysis revealed 2 GLA-gene variants, i.e., p.R356Q and p.G360R. This clinical case highlights that the finding of 2 or more GLA gene variants in a Fabry patient should lead to a careful evaluation in order to determine their exact role in the condition. This case also provides the first clinical evidence that the p.G360R mutation is pathogenic and responsible for a classic phenotype of Fabry disease. The clinical improvement following the initiation of enzyme replacement therapy reinforces the importance of Fabry disease awareness and diagnosis in patients exhibiting red flags, such as left ventricular hypertrophy and stroke.

Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants

2006 ◽  
Vol 290 (4) ◽  
pp. C1076-C1082 ◽  
Author(s):  
Gary Hin-Fai Yam ◽  
Nils Bosshard ◽  
Christian Zuber ◽  
Beat Steinmann ◽  
Jürgen Roth
Keyword(s):  
Enzyme Activity ◽  
Fabry Disease ◽  
Competitive Inhibitor ◽  
Apparent Difference ◽  
Residual Enzyme Activity ◽  
Lysosomal Storage ◽  
Electron Microscopic ◽  
Pharmacological Chaperone ◽  
Mutant Proteins

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A (α-Gal A) resulting in lysosomal accumulation of glycosphingolipid globotriosylceramide Gb3. Misfolded α-Gal A variants can have residual enzyme activity but are unstable. Their lysosomal trafficking is impaired because they are retained in the endoplasmic reticulum (ER) by quality control. Subinhibitory doses of the competitive inhibitor of α-Gal A, 1-deoxygalactonojirimycin (DGJ), stabilize mutant α-Gal A in vitro and correct the trafficking defect. We showed by immunolabeling that the chaperone-like action of DGJ significantly reduces the lysosomal Gb3 storage in human Fabry fibroblasts harboring the novel mutations T194I and V390fsX8. The specificity of the DGJ effect was proven by RNA interference. Electron microscopic morphometry demonstrated a reduction of large-size, disease-associated lysosomes and loss of characteristic multilamellar lysosomal inclusions on DGJ treatment. In addition, the pre-Golgi intermediates were decreased. However, the rough ER was not different between DGJ-treated and untreated cells. Pulse-chase experiments revealed that DGJ treatment resulted in maturation and stabilization of mutant α-Gal A. Genes involved in cell stress signaling, heat shock response, unfolded protein response, and ER-associated degradation show no apparent difference in expression between untreated and DGJ-treated fibroblasts. The DGJ treatment has no apparent cytotoxic effects. Thus our data show the usefulness of a pharmacological chaperone for correction of the lysosomal storage in Fabry fibroblasts harboring different mutations with residual enzyme activity. Pharmacological chaperones acting on misfolded, unstable mutant proteins that exhibit residual biological activity offer a convenient and cost-efficient therapeutic strategy.

Accuracy diagnosis improvement of Fabry disease from dried blood spots: Enzyme activity, lyso‐Gb3 accumulation and GLA gene sequencing

2021 ◽  
Author(s):  
Rocío Delarosa‐Rodríguez ◽  
José D. Santotoribio ◽  
Hernández‐Arévalo Paula ◽  
Antonio González‐Meneses ◽  
Salvador García‐Morillo ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Fabry Disease ◽  
Gene Sequencing ◽  
Dried Blood Spots ◽  
Blood Spots ◽  
Gla Gene

Treatment of Anderson-Fabry Disease

2020 ◽  
Vol 26 (40) ◽  
pp. 5089-5099 ◽  
Author(s):  
Irene Simonetta ◽  
Antonino Tuttolomondo ◽  
Mario Daidone ◽  
Salvatore Miceli ◽  
Antonio Pinto
Keyword(s):  
Fabry Disease ◽  
Treatment Strategies ◽  
Signs And Symptoms ◽  
Current Treatment ◽  
Viral Gene ◽  
Pharmacological Chaperone ◽  
Enzyme Replacement ◽  
Cell Based Therapy ◽  
Organ Manifestations ◽  
Alpha Galactosidase

: Fabry disease is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, predominantly globotriaosylsphingosine (Gb3) in lysosomes, as well as other cellular compartments of several tissues, causing multi-organ manifestations (acroparesthesias, hypohidrosis, angiokeratomas, signs and symptoms of cardiac, renal, cerebrovascular involvement). Pathogenic mutations lead to a deficiency of the lysosomal enzyme alpha-galactosidase A (GLA). In the presence of high clinical suspicion, a careful physical examination and specific laboratory tests are required. Finally, the diagnosis of Fabry’s disease is confirmed by the demonstration of the absence of or reduced alpha-galactosidase A enzyme activity in hemizygous men and gene typing in heterozygous females. Measurement of the biomarkers Gb3 and Lyso Gb3 in biological specimens may facilitate diagnosis. The current treatment of Anderson-Fabry disease is represented by enzyme replacement therapy (ERT) and oral pharmacological chaperone. Future treatments are based on new strategic approaches such as stem cell-based therapy, pharmacological approaches chaperones, mRNA therapy, and viral gene therapy. : This review outlines the current therapeutic approaches and emerging treatment strategies for Anderson-Fabry disease.

scholarly journals PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

2021 ◽  
Vol 11 (9) ◽  
pp. 3729
Author(s):  
Katarzyna Balon ◽  
Benita Wiatrak
Keyword(s):  
Cell Culture ◽  
Dna Damage ◽  
Cell Lines ◽  
Inflammatory Factor ◽  
Research Model ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Primary Cell Lines ◽  
Neurobiological Research ◽  
The Impact

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

scholarly journals Considerations for Home-Based Treatment of Fabry Disease in Poland during the COVID-19 Pandemic and Beyond

2021 ◽  
Vol 18 (16) ◽  
pp. 8242
Author(s):  
Michał Nowicki ◽  
Stanisława Bazan-Socha ◽  
Mariusz Kłopotowski ◽  
Beata Błażejewska-Hyżorek ◽  
Mariusz Kusztal ◽  
...  
Keyword(s):  
Fabry Disease ◽  
Genetic Diseases ◽  
Healthcare Providers ◽  
Treatment Programs ◽  
Current Therapy ◽  
Term Care ◽  
Pharmacological Chaperone ◽  
Enzyme Replacement ◽  
Home Based ◽  
Rare Genetic Diseases

Current therapy for Anderson–Fabry disease in Poland includes hospital or clinic-based intravenous enzyme replacement therapy with recombinant agalsidase alpha or beta, or oral pharmacological chaperone therapy with migalastat. Some countries around the world offer such treatment to patients in the comfort of their own homes. The 2020–2021 COVID-19 pandemic has pushed global healthcare providers to evolve their services so as to minimize the risk of COVID-19 exposure to both patients and providers; this has led to advances in telemedicine services and the increasing availability of at-home treatment for various procedures including parenteral drug administration. A total of 80% of surveyed Anderson–Fabry disease patients in Poland would prefer home-based treatment, which would be a safe and convenient alternative to clinic-based treatment if patient selection is based on our proposed algorithm. Our recommendations for home-based treatments appear feasible for the long term care of Anderson–Fabry disease patients during the COVID-19 pandemic and beyond. This may also serve as a basis for home-based treatment programs in other rare and ultra-rare genetic diseases.

The role of inflammation in recent-onset dilated cardiomyopathy

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Chaloupka ◽  
J Krejci ◽  
H Poloczkova ◽  
P Hude ◽  
E Ozabalova ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Left Ventricular ◽  
Pcr Analysis ◽  
Myocardial Inflammation ◽  
Gene Variants ◽  
Absolute Increase ◽  
Recent Onset ◽  
Cardiotropic Viruses

Abstract Background The aetiology of recent-onset dilated cardiomyopathy (RODCM) includes inflammatory, genetic, toxic and metabolic causes. Delineating the role of inflammation on the genetic background could improve risk stratification. Purpose We aimed to ascertain the role of inflammation evaluated by serum CRP immunohistochemical and PCR analysis of endomyocardial biopsy (EMB) in conjunction with genetic testing in left ventricular reverse remodelling (LVRR) in 12-month follow-up. Methods 83 RODCM patients enrolled in this prospective observational study underwent 12-month echocardiographic follow up whole-exome sequencing, and EMB. Presence of cardiotropic viruses was determined by PCR analysis of the EMB samples. Inflammation was defined according to TIMIC immunohistochemical criteria as the presence of >7 CD3+ lymphocytes/mm2 and/or >14 infiltrating leukocytes (LCA+ cells/mm2). LVRR was defined as an absolute increase in LV ejection fraction > +10% and a relative decrease of LV end-diastolic diameter >−10% at 12 months. Results LVRR occurred in 28 (34%) of all cases. PCR analysis uncovered cardiotropic viruses in 55 (66%) patients, with highest prevalence of parvovirus B19 (47%). (Figure 1) EMB analysis detected inflammation in 28 (34%) cases and inflammation significantly positively predicted LVRR (P=0.019). Sequencing identified disease-related gene variants (ACMG class 3–5) in 45 (54%) patients. Carriers of non-titin gene variants showed a lowest probability of 12-month LVRR (19%) P=0.041. Combination of genetic findings and inflammation did not improve the prediction of LVRR in 12 months. (Table 1) Conclusion Both myocardial inflammation and disease-causing variants can be identified in a large proportion of RODCM cases. Prognostic value of CRP and virus detection is low. Non-titin disease-related variants carriers of are less likely to reach LVRR. In contrast, myocardial inflammation detected by EMB predicts favourable remodelling in 12 months. Figure 1 Funding Acknowledgement Type of funding source: None

scholarly journals Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Dominique Lison ◽  
Saloua Ibouraadaten ◽  
Sybille van den Brule ◽  
Milica Todea ◽  
Adriana Vulpoi ◽  
...  
Keyword(s):  
Carbon Nanotubes ◽  
Cell Culture ◽  
Pulsed Laser ◽  
Lung Fibroblasts ◽  
Laser Microscopy ◽  
Cell Responses ◽  
The Impact ◽  
Delivered Dose ◽  
Femtosecond Pulsed Laser

Abstract Background In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). Methods and results We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1β by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1β responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. Conclusion The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

Synthesis of multimeric pyrrolidine iminosugar inhibitors of human β-glucocerebrosidase and α-galactosidase A: First example of a multivalent enzyme activity enhancer for Fabry disease

2020 ◽  
Vol 192 ◽  
pp. 112173
Author(s):  
Macarena Martínez-Bailén ◽  
Ana T. Carmona ◽  
Francesca Cardona ◽  
Camilla Matassini ◽  
Andrea Goti ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Fabry Disease

scholarly journals Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology

2016 ◽  
Vol 311 (5) ◽  
pp. F1015-F1024 ◽  
Author(s):  
Ester M. Pereira ◽  
Anatália Labilloy ◽  
Megan L. Eshbach ◽  
Ankita Roy ◽  
Arohan R. Subramanya ◽  
...  
Keyword(s):  
Fabry Disease ◽  
Human Kidney ◽  
Premature Death ◽  
Antibody Array ◽  
Cell Models ◽  
Lysosomal Storage ◽  
Protein Levels ◽  
Gla Gene ◽  
Fabry Nephropathy ◽  
And Control

Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable α-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.

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