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Abstracts / Molecular Genetics and Metabolism 117 (2016) S14–S124
of rapid desensitization in 413 cases. J Allergy Clin Immunol 2008: 122: 5. Ensina LF et al. Laronidase hypersensitivity and desensitization in type I mucopolysaccharidosis: a case report. Pediatr Allergy Immunol 2014 Aug;25(5):498-9. doi:10.1016/j.ymgme.2015.12.181
24 Infusion related hypersensitivity reaction to enzyme replacement therapy for lysosomal diseases Carolina Aranda, Luis Felipe Ensina, Carmen Mendes, Ines CameloNunes, Marco Curiati, Marcia Mallozi, Dirceu Solé, Ana Maria Martins, Universidade Federal de São Paulo, São Paulo, Brazil Enzyme replacement therapy (ERT) has been used in the treatment of lysosomal diseases (LD). Infusion-related reactions (IRR) to ERT can occur and can be severe, precluding further ERT. This study was aimed at standardizing the management of IRR. Patients under ERT for mucopolysaccharidosis (I, II and VI), Gaucher, Fabry and Pompe diseases were followed at Federal University of São Paulo, from January 2011 through December 2014. In the presence of signs consistent with adverse reaction, ERT was stopped and skin tests (ST) for specific IgE assay were performed. In patients with symptoms of acute infection after infusion and with negative ST, ERT was maintained at the same infusion rate. For patients with no history of infection and negative ST, ERT was continued at a slower infusion rate. Patients with positive ST, a rapid 3 bags 12-steps desensitization protocol was generated. Among seventy-five LD patients receiving ERT, 12 (16%) developed adverse reactions during infusion. Urticaria was the most prevalent event (40%), followed by fever (20%), chills (20%), cough (13%) and anaphylaxis (13%). Five patients (42%) had positive skin tests. Of these patients, four were successfully underwent desensitization. The mechanism mediated by IgE is the most common in allergic reactions. Cytokine release syndrome can involve complement components. The infected patient may exhibit adverse reactions to some drugs because of preactivated T cells. The standard prevention is based in prophylactic use of antihistamines and corticosteroids. Immune modulation consists of the suppression of B lymphocytes. Desensitization is the process of inducing a state of temporary tolerance in a patient sensitized to a drug through sequential exposure of increasing doses of the drug. Considering the importance of ERT in reducing LD morbidity and mortality, it is critical to recognize the early signs of IRR, understand their mechanism and their management including desensitization. doi:10.1016/j.ymgme.2015.12.182
25 Simultaneous analysis of glucosylceramide and galactosylceramide isoforms in mouse and human brain tissue samples using UPLC-MS/MS Christiane Auray-Blaisa, Michel Boutina, John J. Shackab, aUniversité de Sherbrooke, Sherbrooke, QC, Canada, bUniversity of Alabama at Birmingham, Birmingham, AL, United States Mutations in the GBA gene causing glucocerebrosidase (GCase) deficiency lead to Gaucher disease, and represent a major risk factor for Parkinson disease (PD). Deficient GCase enzymatic activity results in the accumulation of glucosylceramide (GluCer), the main substrate, in biological fluids and tissues. GluCer presents different isoforms owing to different fatty acid moieties. A method based on liquid
chromatography-tandem mass spectrometry was developed for the analysis of GluCer in human and murine brain tissue samples. The main issue was to separate GluCer from its isobaric interference galactosylceramide (GalCer) found in high levels. These two structural isomers may be differentiated only by the axial or equatorial conformation of one hydroxyl group. The devised method was used to analyze brain tissue samples from wild-type mice, Gaucher mice, human controls and PD patients. Mouse or human brain tissue samples were homogenized in methanol using a bead mill (Bead Ruptor 12, Omni). Brain homogenates were extracted using a hydrophiliclipophilic balance (HLB) solid phase extraction cartridges (Oasis, Waters). Samples were separated by ultra-performance liquid chromatography (UPLC) on an Acquity I-Class system (Waters) using a normal phase column (Hilic). A Xevo TQ-S tandem mass spectrometer (Waters) was used in the Multiple Reaction Monitoring (MRM) mode for the detection. Five GluCer isoforms (C18:0, C20:0, C22:0, C24:1, C24:0) were analyzed. Synthetic GalCer(C15:0) was used for the calibration curve, and GluCer(C16:0)D3 as the internal standard. Significantly higher levels of GluCer isoforms were measured in brain tissues from Gaucher mice compared to wild-type mice. However, no statistically significant differences were observed between GluCer isoform levels in human PD brain samples compared to human control brain samples. A technological transfer of this UPLC-MS/MS methodology will be done for the analysis of GluCer in tissues of the murine model of Gaucher disease following different treatments, as well as for Gaucher disease patients. Acknowledgements: Michael J Fox Foundation. doi:10.1016/j.ymgme.2015.12.183
26 Three cases of familial pseudodominance in Pompe disease: Are current practices missing diagnostic and treatment opportunities? Stephanie Austina, Lauren Baileya, Jennifer Sullivana, Carrie Baileyb, David Viskochillb, Priya Kishnania, aDuke University Medical Center, Durham, NC, United States, bUniversity of Utah, Salt Lake City, UT, United States Pompe disease (glycogen storage disease type II) has a broad spectrum of phenotype ranging from the classic infantile presentation with severe hypotonia and cardiomyopathy to a late onset form with a limb girdle muscular dystrophy and respiratory insufficiency. The US Food and Drug Adminstration approved alglucosidase alfa enzyme replacement therapy (ERT) for Pompe disease in 2006. Clinical studies indicate that treatment with ERT results in improved clinical outcomes. We present three non-consanguineous families with multigenerational, phenotypically variable Pompe disease. Case 1: In 2014 a proband presented with molecularly confirmed infantile Pompe disease. In 2015 her mother was molecularly confirmed to have late onset Pompe Disease (LOPD). Testing in the proband’s full siblings determined that two, ages 4 and 6 years have LOPD. Case 2: A proband was enzymatically confirmed to have infantile onset Pompe in 1997. In 2008, her mother was molecularly confirmed to have LOPD. Case 3: In 1994, an adult sib-pair were enzymatically confirmed to have LOPD. In 2002, a first cousin twice removed presented with infantile disease. Molecular testing confirmed a common disease allele shared by these family members. Three additional cases of pseudo dominance in Pompe disease have been published previously - one parent-child pair and two grandparentchild pairs. In 2015, the United States Secretary of Health and Human Services added Pompe disease as a core condition to the Recommended Uniform Screening Panel for state newborn screening (NBS). Published data from NBS programs in Taiwan, Austria, and the state
Abstracts / Molecular Genetics and Metabolism 117 (2016) S14–S124
of rapid desensitization in 413 cases. J Allergy Clin Immunol 2008: 122: 5. Ensina LF et al. Laronidase hypersensitivity and desensitization in type I mucopolysaccharidosis: a case report. Pediatr Allergy Immunol 2014 Aug;25(5):498-9. doi:10.1016/j.ymgme.2015.12.181
24 Infusion related hypersensitivity reaction to enzyme replacement therapy for lysosomal diseases Carolina Aranda, Luis Felipe Ensina, Carmen Mendes, Ines CameloNunes, Marco Curiati, Marcia Mallozi, Dirceu Solé, Ana Maria Martins, Universidade Federal de São Paulo, São Paulo, Brazil Enzyme replacement therapy (ERT) has been used in the treatment of lysosomal diseases (LD). Infusion-related reactions (IRR) to ERT can occur and can be severe, precluding further ERT. This study was aimed at standardizing the management of IRR. Patients under ERT for mucopolysaccharidosis (I, II and VI), Gaucher, Fabry and Pompe diseases were followed at Federal University of São Paulo, from January 2011 through December 2014. In the presence of signs consistent with adverse reaction, ERT was stopped and skin tests (ST) for specific IgE assay were performed. In patients with symptoms of acute infection after infusion and with negative ST, ERT was maintained at the same infusion rate. For patients with no history of infection and negative ST, ERT was continued at a slower infusion rate. Patients with positive ST, a rapid 3 bags 12-steps desensitization protocol was generated. Among seventy-five LD patients receiving ERT, 12 (16%) developed adverse reactions during infusion. Urticaria was the most prevalent event (40%), followed by fever (20%), chills (20%), cough (13%) and anaphylaxis (13%). Five patients (42%) had positive skin tests. Of these patients, four were successfully underwent desensitization. The mechanism mediated by IgE is the most common in allergic reactions. Cytokine release syndrome can involve complement components. The infected patient may exhibit adverse reactions to some drugs because of preactivated T cells. The standard prevention is based in prophylactic use of antihistamines and corticosteroids. Immune modulation consists of the suppression of B lymphocytes. Desensitization is the process of inducing a state of temporary tolerance in a patient sensitized to a drug through sequential exposure of increasing doses of the drug. Considering the importance of ERT in reducing LD morbidity and mortality, it is critical to recognize the early signs of IRR, understand their mechanism and their management including desensitization. doi:10.1016/j.ymgme.2015.12.182
25 Simultaneous analysis of glucosylceramide and galactosylceramide isoforms in mouse and human brain tissue samples using UPLC-MS/MS Christiane Auray-Blaisa, Michel Boutina, John J. Shackab, aUniversité de Sherbrooke, Sherbrooke, QC, Canada, bUniversity of Alabama at Birmingham, Birmingham, AL, United States Mutations in the GBA gene causing glucocerebrosidase (GCase) deficiency lead to Gaucher disease, and represent a major risk factor for Parkinson disease (PD). Deficient GCase enzymatic activity results in the accumulation of glucosylceramide (GluCer), the main substrate, in biological fluids and tissues. GluCer presents different isoforms owing to different fatty acid moieties. A method based on liquid
chromatography-tandem mass spectrometry was developed for the analysis of GluCer in human and murine brain tissue samples. The main issue was to separate GluCer from its isobaric interference galactosylceramide (GalCer) found in high levels. These two structural isomers may be differentiated only by the axial or equatorial conformation of one hydroxyl group. The devised method was used to analyze brain tissue samples from wild-type mice, Gaucher mice, human controls and PD patients. Mouse or human brain tissue samples were homogenized in methanol using a bead mill (Bead Ruptor 12, Omni). Brain homogenates were extracted using a hydrophiliclipophilic balance (HLB) solid phase extraction cartridges (Oasis, Waters). Samples were separated by ultra-performance liquid chromatography (UPLC) on an Acquity I-Class system (Waters) using a normal phase column (Hilic). A Xevo TQ-S tandem mass spectrometer (Waters) was used in the Multiple Reaction Monitoring (MRM) mode for the detection. Five GluCer isoforms (C18:0, C20:0, C22:0, C24:1, C24:0) were analyzed. Synthetic GalCer(C15:0) was used for the calibration curve, and GluCer(C16:0)D3 as the internal standard. Significantly higher levels of GluCer isoforms were measured in brain tissues from Gaucher mice compared to wild-type mice. However, no statistically significant differences were observed between GluCer isoform levels in human PD brain samples compared to human control brain samples. A technological transfer of this UPLC-MS/MS methodology will be done for the analysis of GluCer in tissues of the murine model of Gaucher disease following different treatments, as well as for Gaucher disease patients. Acknowledgements: Michael J Fox Foundation. doi:10.1016/j.ymgme.2015.12.183
26 Three cases of familial pseudodominance in Pompe disease: Are current practices missing diagnostic and treatment opportunities? Stephanie Austina, Lauren Baileya, Jennifer Sullivana, Carrie Baileyb, David Viskochillb, Priya Kishnania, aDuke University Medical Center, Durham, NC, United States, bUniversity of Utah, Salt Lake City, UT, United States Pompe disease (glycogen storage disease type II) has a broad spectrum of phenotype ranging from the classic infantile presentation with severe hypotonia and cardiomyopathy to a late onset form with a limb girdle muscular dystrophy and respiratory insufficiency. The US Food and Drug Adminstration approved alglucosidase alfa enzyme replacement therapy (ERT) for Pompe disease in 2006. Clinical studies indicate that treatment with ERT results in improved clinical outcomes. We present three non-consanguineous families with multigenerational, phenotypically variable Pompe disease. Case 1: In 2014 a proband presented with molecularly confirmed infantile Pompe disease. In 2015 her mother was molecularly confirmed to have late onset Pompe Disease (LOPD). Testing in the proband’s full siblings determined that two, ages 4 and 6 years have LOPD. Case 2: A proband was enzymatically confirmed to have infantile onset Pompe in 1997. In 2008, her mother was molecularly confirmed to have LOPD. Case 3: In 1994, an adult sib-pair were enzymatically confirmed to have LOPD. In 2002, a first cousin twice removed presented with infantile disease. Molecular testing confirmed a common disease allele shared by these family members. Three additional cases of pseudo dominance in Pompe disease have been published previously - one parent-child pair and two grandparentchild pairs. In 2015, the United States Secretary of Health and Human Services added Pompe disease as a core condition to the Recommended Uniform Screening Panel for state newborn screening (NBS). Published data from NBS programs in Taiwan, Austria, and the state