- Email: [email protected]
Behavioural aspects of Gaucher's disease – An explorative study
S28
Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
psychosine. Using the specific fluorescent substrate, 6-hexadecanoylamino-4-methylumbelliferyl-β-D-galactoside(HMU Gal), primary skin fibroblast from patients with different missense mutations showed measurable residual GALC activity when cultured and assayed in 96-well plates. Wild-type(WT)-GALC activity from controls ranges 6-7-fold above the GALC mutants found in patient lines. When miniaturizing to a 384-well plate format, SV-40 transformed skin fibroblasts from patients and controls showed detectable and reproducible GALC activity. The plate uniformity assessment utilizing WT-GALC(CV 0.38%) control and G270D-GALC(CV 0.76%) lines generated a Z’ factor of 0.58. Once developed and validated, the cell-based HTS assay will allow the identification of SMs that enhance residual GALC activity not only by direct interactions, but also by indirect mechanisms, which ultimately have a broader application for treatment of other LSDs.
currently considered for LSDs (www.nbstrn.org). The goal of this project is to collect data sets of at least 50 true positive cases per LSD to establish disease ranges that will allow provision of clinically validated cutoffs and ultimately optimize analytical performance (high sensitivity and positive predictive value, low false positive rate). To achieve this goal, participation by screening laboratories and LSD specialists around the WORLD is crucial. National and international participants will be provided with a user ID and password to gain access to the secure website. Participants have access to pages unique to their NBS program for data submission and comparison tools and to common pages inclusive of project tools and reports. NBS programs are encouraged to contribute screening results of confirmed true positive cases, percentiles of all markers in the respective normal population, cutoff values, performance metrics, and information about the screening assay(s) used. LSD specialists are encouraged to facilitate the retrieval of any residual NBS samples of their patients.
doi:10.1016/j.ymgme.2010.11.092 doi:10.1016/j.ymgme.2010.11.094 Changes in neuropsychological functioning in three pediatric patients with Niemann-Pick disease type C (NPC) following treatment with miglustat
A phase 1 trial of recombinant human acid Sphingomyelinase (rhASM) Enzyme replacement therapy in adults with Non-Neuronopathic ASM deficiency (ASMD Niemann-Pick B)
Eva Mamak, Hospital for Sick Children, Toronto, ON, Canada Niemann-Pick Disease Type C is a rare, neurodegenerative disorder associated with accumulation of cholesterol and glycopphingolipids within late endosomes and lysosomes. Miglustat has been documented to improve eye movement, swallowing, ambulation, and motion in patients with this condition. In addition, neuropsychological functioning in untreated adults has documented impairment in fine motor skills and verbal working memory, but limited information is available regarding pediatric neuropsychological functioning. A case series of three patients (initially evaluated at 4 yrs 2 mos, 4 yrs 0 mos, and 5 yrs 7 mos.) is presented. Diagnosis occurred at 3 months, 2 years, and 3 months, respectively. All three patients received neuropsychological testing at baseline (prior to initiation of miglustat), and one year later. Baseline results indicated variable neuropsychological presentation, ranging from intellectual impairment to average cognitive and adaptive functioning; with marked difficulties in behavioral regulation, fine motor skills, and expressive language. One year later, a pattern of stabilized visual/motor skills and cognitive abilities was noted, with gains in language. Overall, changes in functioning following treatment with miglustat seem most prominent in communication skills. doi:10.1016/j.ymgme.2010.11.093
Pursuing objective performance metrics of Newborn Screening (NBS) tests for LSDs Dietrich Materna, Joseph Orsinib, Neil Maffita, Giselle Bentz Pinoa, Elyse Gryckia, Silvia Tortorellia, Devin Oglesbeea, Dimitar Gavrilova, Piero Rinaldoa, Kimiyo Raymonda, aMayo Clinic College of Medicine, Rochester, MN, USA, bWadsworth Center, New York State Department of Health, Albany, NY, USA NBS for Krabbe disease and/or other LSDs has been implemented in New York, Illinois, and Taiwan. Testing is currently done by enzyme assays using either tandem mass spectrometry or fluorometry. Assays measuring the concentration of relevant proteins or glycosaminoglycans have also been proposed. To prevent a recurrence of the significant variability in NBS performance that characterized the implementation of tandem mass spectrometry into NBS programs, a web site has been created to provide reference and disease ranges for all NBS assays
Margaret McGoverna, Melissa Wassersteinb, Brian Kirmseb, Lane Duvallb, Thomas Schianob, Beth Thurbergc, Susan Richardsc, Gerald Coxc, aStony Brook University School of Medicine, Stony Brook, NY, USA, b Mount Sinai School of Medicine, NY, USA, cGenzyme Corporation, Cambridge, USA Preclinical studies have demonstrated a novel rhASM-associated toxicity (cytokine elevations, shock, and death) observed only in ASM knockout mice following high initial doses (≥10 mg/kg) that appears to be due to rapid sphingomyelin breakdown. The objectives of this Phase 1 trial were to evaluate the safety and pharmacokinetics of rhASM in non-neuronopathic ASMD. Eleven adult patients (mean age 30.5 yr, 6 males, all Caucasian) meeting entry criteria were administered single ascending doses of intravenous rhASM in 5 dose cohorts (0.03, 0.1, 0.3, 0.6, and 1.0 mg/kg). No clinically significant cardiovascular changes occurred. At doses ≥ 0.3 mg/kg, 4 of 5 patients experienced a total of 23 drug-related adverse events. Dose-related rises in ceramide, bilirubin (predominantly indirect), C-reactive protein, cytokines, and other acute phase reactants peaked at 2448 hr post-dose and resolved by Days 3-14. Drug-related clinical symptoms were mild or moderate and included fever (n = 2), pain (myalgia; abdomen, extremity, and hip pain), fatigue, nausea (n = 2), and vomiting. Symptoms began 12 hr post- dose and most were resolved by 72 hr. Day 14 liver biopsies were unchanged from baseline except in 1 patient (cohort 4) with 2 new small inflammatory foci, 1 of which was associated with hepatocellular degeneration. The circulating t1/2 of rhASM was 10-15 hr. In this firstin-patient study of rhASM, the major safety findings were doserelated hyperbilirubinemia, acute phase response, and constitutional symptoms occurring 1-3 days post-dose. The maximum tolerated starting dose was 0.6 mg/kg. Within-patient dose escalation may be an option for higher repeat doses of rhASM. doi:10.1016/j.ymgme.2010.11.095
Behavioural aspects of Gaucher's disease – An explorative study Lauren McPartlana, Ruksana Ahmeda, Ashok Vellodiab, Paramala Santosha, aGreat Ormond Street Hospital, London, Greater London, U.K., bInstitute of Child Health, U.K.
Q60 Q21
Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
Introduction: The management of Gaucher Disease has been revolutionized by enzyme replacement therapy. Consequently, life expectancy has increased. As a result, other features of GD are being ‘uncovered’ and attention is now turning to the neurobehavioural problems. This study aimed to assess the behavioural symptoms displayed by children with GD3. Description/cases/results: The study was a postal and internetbased survey of a group of 7 children with GD3. The subjects were recruited from the Metabolic Clinic at Great Ormond Street Hospital. The parents of the participants completed the Profile of Neuropsychiatric Symptoms questionnaire (PONS) and the parental stress index (PSI).The GD3 patients PONS data showed that compared to the general child population, 71.4% of the patients had difficulties with learning and clumsiness, 42.9% had difficulties arising from sensory symptoms, low mood, manic episodes and oppositional and defiant behaviour and 28.6% had difficulties due to hyperactivity, impulsivity, aggression, poor language, circumscribed interests, selfinjury, poor empathy, obsessions & compulsions, depressive thoughts, worries, fears and poor memory.The PSI highlighted that 50% of GD3 parent's show levels of distress that warrant professional attention. Conclusion/Discussion: These preliminary findings indicate that patients with GD3 do present with behavioural and emotional difficulties. The clinical implications for social functioning, and, crucially, for education, are profound. Larger cohorts need to be studied both in order to corroborate these findings as well as to develop a more detailed behavioural phenotype of GD subtypes. Through a more comprehensive understanding of patients’ psychosocial and psychopharmacological needs, appropriate interventions can be designed.
S29
MRI Findings in Anderson- Fabry disease(AFD) Carmen Mendes, Maria Lucia Borri, Patricia Feliciano, Sandra Kyosen, Carolina Aranda, Maret Rand, Ana Maria Martins, Centro de Referencia em Erros INatos do Metabolismo UNIFESP, Sao Paulo, Sao Paulo, Brazil Background: Anderson – Fabry disease is an X- linked lysossomal storage disorder caused by deficiency of alpha- galactosidase A. As a consequence , there is accumulation of glycosphingolipids in blood vessels , central and peripheral autonomic nervous system. Brain MRI are often abnormal. Methods: The aim of the study was to describe brain MRI data of 10 patients with AFD , 7 male patients and 3 female patients, with age 12 to 60 years old (median age 34,7 ).The brain MRI were analysed retrospectively . Seven patients were under treatment with enzyme replacement therapy ( ERT ). Results: Brain MRI were abnormal in all patients .The most frequently finding was Virchow-Robin spaces enlargement in 6 patients,mild cortical atrophy in 5 ,calcification or metalic deposits in deep gray matter , cerebelar atrophy and microangiopathy in 2 , hipersinal in FLAIR/T2 in pulvinar in 3 patients ,white matter lesion in 4 , enlargement of lateral ventricles in 3 ,microcalcifications in left thalamus,hipersinal in T1 in pulvinar ,and hipersinal in T2 in medium cerebelar peduncle in 1 patient. Conclusions: Neuroradiological findings in AFD include vascular , white matter lesions and increased signal intensity of the pulvinar on T1-weighted images. Only one patient presented characteristic T1 pulvinar sign , and no ischemic strokes was presented in our patients. doi:10.1016/j.ymgme.2010.11.098
doi:10.1016/j.ymgme.2010.11.096
Enhanced Gb3 reduction mediated by Lentivector Transduction of human CD34+ bone marrow-derived cells in a Novel Fabry/NOD/ SCID Xenograft model
Q22
Jeffrey Medin, Natalia Pacienza, Nobuo Mizue, Xin Fan, Matthew Scaife, University Health Network, Toronto, ON, Canada Through 13 generations of a ‘speed congenic’ breeding schema, we have produced a novel Fabry/NOD/SCID mouse model. Genotyping confirmed the loss of the α-galactosidase A (α-gal A) alleles and maintenance of the NOD/SCID mutations. These α-gal A deficient animals have minimal T cells and B cells as measured by flow cytometry and accept human hematopoietic cells as xenografts. Transplantation of normal human hematopoietic cells (specifically CD34+ cells originating from the bone marrow of normal donors) led to Gb3 reductions in numerous organs in these mice; further, overexpression of α-gal A by lentivector (LV)-mediated transduction of such foundation cells prior to infusion dramatically enhanced outcomes. This confirms that metabolic co-operativity or ‘crosscorrection’ effected by such strategies as gene therapy have additional systemic benefits beyond just correction of lipid accumulation in key source cell populations such as hematopoietic cells. This overarching phenomenon drives our efforts to undertake a gene therapy trial for Fabry disease using LVs and targeting this population of cells. Having such a ‘pure’ line of Fabry mice also facilitates genomic/proteomic studies and experiments on lipid accumulation without the confounding issues of strain heterogeneity and lymphocyte contribution; such studies are underway in our laboratory.
doi:10.1016/j.ymgme.2010.11.097
Analysis of Glycosaminoglycans in cerebrospinal fluid using tandem mass spectrometry: Potential for therapeutic monitoring of patients with Mucopolysaccharidoses David Millingtona, Haoyue Zhanga, Sarah Younga, Paul Orchardb, Jakub Tolarb, Christiane Auray-Blaisc, aDuke University Medical Center, Durham, NC, USA, bUniversity of Minnesota, Minneapolis, USA, cUniversity of Sherbrooke, Sherbrooke, Canada Introduction: Mucopolysaccharidoses (MPSs) are complex storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, brain and other tissues. New treatment options for MPS-I and MPS-II that target the brain require new techniques to evaluate their biochemical impact. Accordingly, we developed a tandem mass spectrometry methodology to quantify the GAGs, dermatan sulfate (DS) and heparan sulfate (HS), in small volumes of cerebrospinal fluid (CSF). Method: CSF samples (25 μL) were evaporated under nitrogen and the residues treated with 0.2 mL of 3 M-HCl in methanol for 75 min at 65 °C. After evaporation of the reagent, the methanolysate was mixed with a deuterium-labeled DS and HS lysate mixture in acetonitrile (0.1 mL) that provided isotope-labeled internal standards for the target analytes. Samples were injected into a Acquity® – Xevo-TQD® UPLC-MS/MS system (Waters Corporation) equipped with a BEH® Amide UPLC column (2.1 mm × 50 mm, 1.7 μM particle size) operating under a gradient and the target analytes were detected by selected reaction monitoring in positive ion electrospray mode. Results: CSF from normal pediatric patients contained 0.6 ± 0.2 μg/ mL chondroitin sulfate; ≤0.2 μg/mL DS and undetectable HS. Patients with MPS-I (n = 7) and MPS-II (n = 3) had pre-treatment levels of DS at 1.1-2.5 μg/mL and HS at 3-11 μg/mL. Following intrathecal enzyme replacement therapy (IT-ERT), levels of DS and HS in CSF were significantly reduced.
Q49
Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
psychosine. Using the specific fluorescent substrate, 6-hexadecanoylamino-4-methylumbelliferyl-β-D-galactoside(HMU Gal), primary skin fibroblast from patients with different missense mutations showed measurable residual GALC activity when cultured and assayed in 96-well plates. Wild-type(WT)-GALC activity from controls ranges 6-7-fold above the GALC mutants found in patient lines. When miniaturizing to a 384-well plate format, SV-40 transformed skin fibroblasts from patients and controls showed detectable and reproducible GALC activity. The plate uniformity assessment utilizing WT-GALC(CV 0.38%) control and G270D-GALC(CV 0.76%) lines generated a Z’ factor of 0.58. Once developed and validated, the cell-based HTS assay will allow the identification of SMs that enhance residual GALC activity not only by direct interactions, but also by indirect mechanisms, which ultimately have a broader application for treatment of other LSDs.
currently considered for LSDs (www.nbstrn.org). The goal of this project is to collect data sets of at least 50 true positive cases per LSD to establish disease ranges that will allow provision of clinically validated cutoffs and ultimately optimize analytical performance (high sensitivity and positive predictive value, low false positive rate). To achieve this goal, participation by screening laboratories and LSD specialists around the WORLD is crucial. National and international participants will be provided with a user ID and password to gain access to the secure website. Participants have access to pages unique to their NBS program for data submission and comparison tools and to common pages inclusive of project tools and reports. NBS programs are encouraged to contribute screening results of confirmed true positive cases, percentiles of all markers in the respective normal population, cutoff values, performance metrics, and information about the screening assay(s) used. LSD specialists are encouraged to facilitate the retrieval of any residual NBS samples of their patients.
doi:10.1016/j.ymgme.2010.11.092 doi:10.1016/j.ymgme.2010.11.094 Changes in neuropsychological functioning in three pediatric patients with Niemann-Pick disease type C (NPC) following treatment with miglustat
A phase 1 trial of recombinant human acid Sphingomyelinase (rhASM) Enzyme replacement therapy in adults with Non-Neuronopathic ASM deficiency (ASMD Niemann-Pick B)
Eva Mamak, Hospital for Sick Children, Toronto, ON, Canada Niemann-Pick Disease Type C is a rare, neurodegenerative disorder associated with accumulation of cholesterol and glycopphingolipids within late endosomes and lysosomes. Miglustat has been documented to improve eye movement, swallowing, ambulation, and motion in patients with this condition. In addition, neuropsychological functioning in untreated adults has documented impairment in fine motor skills and verbal working memory, but limited information is available regarding pediatric neuropsychological functioning. A case series of three patients (initially evaluated at 4 yrs 2 mos, 4 yrs 0 mos, and 5 yrs 7 mos.) is presented. Diagnosis occurred at 3 months, 2 years, and 3 months, respectively. All three patients received neuropsychological testing at baseline (prior to initiation of miglustat), and one year later. Baseline results indicated variable neuropsychological presentation, ranging from intellectual impairment to average cognitive and adaptive functioning; with marked difficulties in behavioral regulation, fine motor skills, and expressive language. One year later, a pattern of stabilized visual/motor skills and cognitive abilities was noted, with gains in language. Overall, changes in functioning following treatment with miglustat seem most prominent in communication skills. doi:10.1016/j.ymgme.2010.11.093
Pursuing objective performance metrics of Newborn Screening (NBS) tests for LSDs Dietrich Materna, Joseph Orsinib, Neil Maffita, Giselle Bentz Pinoa, Elyse Gryckia, Silvia Tortorellia, Devin Oglesbeea, Dimitar Gavrilova, Piero Rinaldoa, Kimiyo Raymonda, aMayo Clinic College of Medicine, Rochester, MN, USA, bWadsworth Center, New York State Department of Health, Albany, NY, USA NBS for Krabbe disease and/or other LSDs has been implemented in New York, Illinois, and Taiwan. Testing is currently done by enzyme assays using either tandem mass spectrometry or fluorometry. Assays measuring the concentration of relevant proteins or glycosaminoglycans have also been proposed. To prevent a recurrence of the significant variability in NBS performance that characterized the implementation of tandem mass spectrometry into NBS programs, a web site has been created to provide reference and disease ranges for all NBS assays
Margaret McGoverna, Melissa Wassersteinb, Brian Kirmseb, Lane Duvallb, Thomas Schianob, Beth Thurbergc, Susan Richardsc, Gerald Coxc, aStony Brook University School of Medicine, Stony Brook, NY, USA, b Mount Sinai School of Medicine, NY, USA, cGenzyme Corporation, Cambridge, USA Preclinical studies have demonstrated a novel rhASM-associated toxicity (cytokine elevations, shock, and death) observed only in ASM knockout mice following high initial doses (≥10 mg/kg) that appears to be due to rapid sphingomyelin breakdown. The objectives of this Phase 1 trial were to evaluate the safety and pharmacokinetics of rhASM in non-neuronopathic ASMD. Eleven adult patients (mean age 30.5 yr, 6 males, all Caucasian) meeting entry criteria were administered single ascending doses of intravenous rhASM in 5 dose cohorts (0.03, 0.1, 0.3, 0.6, and 1.0 mg/kg). No clinically significant cardiovascular changes occurred. At doses ≥ 0.3 mg/kg, 4 of 5 patients experienced a total of 23 drug-related adverse events. Dose-related rises in ceramide, bilirubin (predominantly indirect), C-reactive protein, cytokines, and other acute phase reactants peaked at 2448 hr post-dose and resolved by Days 3-14. Drug-related clinical symptoms were mild or moderate and included fever (n = 2), pain (myalgia; abdomen, extremity, and hip pain), fatigue, nausea (n = 2), and vomiting. Symptoms began 12 hr post- dose and most were resolved by 72 hr. Day 14 liver biopsies were unchanged from baseline except in 1 patient (cohort 4) with 2 new small inflammatory foci, 1 of which was associated with hepatocellular degeneration. The circulating t1/2 of rhASM was 10-15 hr. In this firstin-patient study of rhASM, the major safety findings were doserelated hyperbilirubinemia, acute phase response, and constitutional symptoms occurring 1-3 days post-dose. The maximum tolerated starting dose was 0.6 mg/kg. Within-patient dose escalation may be an option for higher repeat doses of rhASM. doi:10.1016/j.ymgme.2010.11.095
Behavioural aspects of Gaucher's disease – An explorative study Lauren McPartlana, Ruksana Ahmeda, Ashok Vellodiab, Paramala Santosha, aGreat Ormond Street Hospital, London, Greater London, U.K., bInstitute of Child Health, U.K.
Q60 Q21
Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47
Introduction: The management of Gaucher Disease has been revolutionized by enzyme replacement therapy. Consequently, life expectancy has increased. As a result, other features of GD are being ‘uncovered’ and attention is now turning to the neurobehavioural problems. This study aimed to assess the behavioural symptoms displayed by children with GD3. Description/cases/results: The study was a postal and internetbased survey of a group of 7 children with GD3. The subjects were recruited from the Metabolic Clinic at Great Ormond Street Hospital. The parents of the participants completed the Profile of Neuropsychiatric Symptoms questionnaire (PONS) and the parental stress index (PSI).The GD3 patients PONS data showed that compared to the general child population, 71.4% of the patients had difficulties with learning and clumsiness, 42.9% had difficulties arising from sensory symptoms, low mood, manic episodes and oppositional and defiant behaviour and 28.6% had difficulties due to hyperactivity, impulsivity, aggression, poor language, circumscribed interests, selfinjury, poor empathy, obsessions & compulsions, depressive thoughts, worries, fears and poor memory.The PSI highlighted that 50% of GD3 parent's show levels of distress that warrant professional attention. Conclusion/Discussion: These preliminary findings indicate that patients with GD3 do present with behavioural and emotional difficulties. The clinical implications for social functioning, and, crucially, for education, are profound. Larger cohorts need to be studied both in order to corroborate these findings as well as to develop a more detailed behavioural phenotype of GD subtypes. Through a more comprehensive understanding of patients’ psychosocial and psychopharmacological needs, appropriate interventions can be designed.
S29
MRI Findings in Anderson- Fabry disease(AFD) Carmen Mendes, Maria Lucia Borri, Patricia Feliciano, Sandra Kyosen, Carolina Aranda, Maret Rand, Ana Maria Martins, Centro de Referencia em Erros INatos do Metabolismo UNIFESP, Sao Paulo, Sao Paulo, Brazil Background: Anderson – Fabry disease is an X- linked lysossomal storage disorder caused by deficiency of alpha- galactosidase A. As a consequence , there is accumulation of glycosphingolipids in blood vessels , central and peripheral autonomic nervous system. Brain MRI are often abnormal. Methods: The aim of the study was to describe brain MRI data of 10 patients with AFD , 7 male patients and 3 female patients, with age 12 to 60 years old (median age 34,7 ).The brain MRI were analysed retrospectively . Seven patients were under treatment with enzyme replacement therapy ( ERT ). Results: Brain MRI were abnormal in all patients .The most frequently finding was Virchow-Robin spaces enlargement in 6 patients,mild cortical atrophy in 5 ,calcification or metalic deposits in deep gray matter , cerebelar atrophy and microangiopathy in 2 , hipersinal in FLAIR/T2 in pulvinar in 3 patients ,white matter lesion in 4 , enlargement of lateral ventricles in 3 ,microcalcifications in left thalamus,hipersinal in T1 in pulvinar ,and hipersinal in T2 in medium cerebelar peduncle in 1 patient. Conclusions: Neuroradiological findings in AFD include vascular , white matter lesions and increased signal intensity of the pulvinar on T1-weighted images. Only one patient presented characteristic T1 pulvinar sign , and no ischemic strokes was presented in our patients. doi:10.1016/j.ymgme.2010.11.098
doi:10.1016/j.ymgme.2010.11.096
Enhanced Gb3 reduction mediated by Lentivector Transduction of human CD34+ bone marrow-derived cells in a Novel Fabry/NOD/ SCID Xenograft model
Q22
Jeffrey Medin, Natalia Pacienza, Nobuo Mizue, Xin Fan, Matthew Scaife, University Health Network, Toronto, ON, Canada Through 13 generations of a ‘speed congenic’ breeding schema, we have produced a novel Fabry/NOD/SCID mouse model. Genotyping confirmed the loss of the α-galactosidase A (α-gal A) alleles and maintenance of the NOD/SCID mutations. These α-gal A deficient animals have minimal T cells and B cells as measured by flow cytometry and accept human hematopoietic cells as xenografts. Transplantation of normal human hematopoietic cells (specifically CD34+ cells originating from the bone marrow of normal donors) led to Gb3 reductions in numerous organs in these mice; further, overexpression of α-gal A by lentivector (LV)-mediated transduction of such foundation cells prior to infusion dramatically enhanced outcomes. This confirms that metabolic co-operativity or ‘crosscorrection’ effected by such strategies as gene therapy have additional systemic benefits beyond just correction of lipid accumulation in key source cell populations such as hematopoietic cells. This overarching phenomenon drives our efforts to undertake a gene therapy trial for Fabry disease using LVs and targeting this population of cells. Having such a ‘pure’ line of Fabry mice also facilitates genomic/proteomic studies and experiments on lipid accumulation without the confounding issues of strain heterogeneity and lymphocyte contribution; such studies are underway in our laboratory.
doi:10.1016/j.ymgme.2010.11.097
Analysis of Glycosaminoglycans in cerebrospinal fluid using tandem mass spectrometry: Potential for therapeutic monitoring of patients with Mucopolysaccharidoses David Millingtona, Haoyue Zhanga, Sarah Younga, Paul Orchardb, Jakub Tolarb, Christiane Auray-Blaisc, aDuke University Medical Center, Durham, NC, USA, bUniversity of Minnesota, Minneapolis, USA, cUniversity of Sherbrooke, Sherbrooke, Canada Introduction: Mucopolysaccharidoses (MPSs) are complex storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, brain and other tissues. New treatment options for MPS-I and MPS-II that target the brain require new techniques to evaluate their biochemical impact. Accordingly, we developed a tandem mass spectrometry methodology to quantify the GAGs, dermatan sulfate (DS) and heparan sulfate (HS), in small volumes of cerebrospinal fluid (CSF). Method: CSF samples (25 μL) were evaporated under nitrogen and the residues treated with 0.2 mL of 3 M-HCl in methanol for 75 min at 65 °C. After evaporation of the reagent, the methanolysate was mixed with a deuterium-labeled DS and HS lysate mixture in acetonitrile (0.1 mL) that provided isotope-labeled internal standards for the target analytes. Samples were injected into a Acquity® – Xevo-TQD® UPLC-MS/MS system (Waters Corporation) equipped with a BEH® Amide UPLC column (2.1 mm × 50 mm, 1.7 μM particle size) operating under a gradient and the target analytes were detected by selected reaction monitoring in positive ion electrospray mode. Results: CSF from normal pediatric patients contained 0.6 ± 0.2 μg/ mL chondroitin sulfate; ≤0.2 μg/mL DS and undetectable HS. Patients with MPS-I (n = 7) and MPS-II (n = 3) had pre-treatment levels of DS at 1.1-2.5 μg/mL and HS at 3-11 μg/mL. Following intrathecal enzyme replacement therapy (IT-ERT), levels of DS and HS in CSF were significantly reduced.
Q49