- Email: [email protected]
Molecular basis of sialidosis and its treatment
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
GM2 gangliosidoses, including Tay–Sachs disease (TSD) and Sandhoff disease (SD), are neurodegenerative lysosomal diseases (LSD) associated with excessive accumulation of GM2 ganglioside (GM2) in the brain and neurological symptoms. TSD and SD are the lysosomal β-hexosaminidase A (HexA) deficiencies caused by gene mutations in HEXA and HEXB loci, which encode the α- and β-subunit of HexA (αβ heterodimer), respectively. Hence there are no effective treatments for GM2 gangliosidoses, although several therapeutic approaches have been developed including gene therapy, enzyme replacement therapy (ERT), substrate reduction therapy, etc. Previously we constructed a human modified HEXB cDNA encoding altered β-subunit containing partial amino acid sequence of the α-subunit, and succeeded in the production of the modified HexB (modB) by CHO cell line with the GM2-degrading activity in the brain by intracerebroventricular (icv) administration to SD model mice. In this study, we also designed mod2B containing additional α-sequences to resist protease degradation. The recombinant modB and mod2B carrying N-glycans with terminal mannose 6-phosphate (M6P) residues (M6P-modB and -mod2B) were purified with three-step column chromatography, including an affinity chromatography with Phos-Tag resin, which is usually used to capture phosphorylated proteins. Icv injection of M6P-modB caused uptake by parenchymal neural cells, which was processed to the mature form and partially degraded Hex subunits. In contrast, M6P-mod2B was resistant to neural proteases and maintained the mature form and the GM2-degrading activity. These results suggest that α-sequence around GSEP loop should regulate the resistance to neural proteases. Next, we visualized the delivery of M6P-modB and -mod2B to lysosomes in the target cells derived from TSD and SD model through binding with cell surface cation-independent M6P receptors (CI-M6PR) with novel acidic pHactivatable fluorescent probes, including RhPM as a tag conjugated to modified Hex proteins. We also developed an imaging mass spectrometry (IMS) system to evaluate the distribution and reduction of GM2 and asialo GM2 (GA2) accumulated in the brain sections of SD mice on icv administration. In the brains of untreated SD mice, signals of GM2 (d18:1/18:0) were prominent in the hypothalamus (HTH) and lateral septal nucleus (LSN). GM2 (d18:1/20:0) were observed especially, in the cerebellum (CB). Signals of GA2 (d18:1/18:0) and GA2 (d18:1/20:0) were observed throughout the brain. On treatment with M6P-mod2B, compared to M6P-modB, signals of GM2 and GA2 were remarkably decreased in the whole area including cerebral cortex (CCX), CB, HTH and LSN. Furthermore, icv administration of M6P-mod2B has remarkable therapeutic effects on SD mice, including improvement of motor dysfunction. These results suggest that novel intracerebroventricular ERT with protease-resistant modified human β-hexosaminidase B (M6Pmod2B) should be applicable for patients with GM2 gangliosidoses.
UN
CO
RR
EC
TE
DP
Background: In 2014, the Pompe Registry is recognizing and celebrating an important milestone: the 10-year anniversary of the Registry. Methods: Started in 2004 and sponsored by Genzyme, a Sanofi company, the Pompe Registry was established as a global, voluntary, observational database to collect and report on the natural history and outcomes of patients with Pompe disease through routine clinical practice, regardless of patient treatment status. Data currently are collected and reported by enrolling clinicians worldwide in four geographic regions: Europe, North America, Latin America, and the Asia Pacific region. Results: As of May 2014, 1292 patients in 34 countries were enrolled in the Registry. The majority (70.1%) was Caucasian, and there was an approximate equal distribution of males and females. More than three-quarters (78.9%) of enrolled patients had Late-onset Pompe disease (LOPD), defined as symptom onset ≤12 months of age without cardiomyopathy or symptom onset N12 months of age; 13.8% of patients had classic Infantile-onset Pompe disease (IOPD), defined as symptom onset ≤12 months with cardiomyopathy. Age at symptom onset was unknown in 7.3% of patients. In classic IOPD, the overall mean age at symptom onset was 2.9 months in 2014. The mean age at diagnosis for these patients was 4.0 months, signifying a diagnostic gap of 1.1 months. In 2014, the overall mean age of symptom onset and diagnosis for LOPD patients was 26.5 years and 33.5 years, respectively. Blood-based enzyme assays that measure acid alpha-glucosidase (GAA) activity (including dried blood spots [DBS], leukocytes, and lymphocytes), and DNA analysis were the most frequently used methods of diagnosis for patients in the Pompe Registry. Among classic IOPD patients, hypotonia was the most frequently reported symptom overall, affecting 94.7% of patients. Ambulation was impaired in 78.4% of LOPD patients in the Registry. Two-thirds (67.6%) of LOPD patients reported to have lost ambulatory capabilities, and approximately half reported using ambulation devices. Overall, 47.1% of all Registry patients had reported receiving respiratory support of some kind: 43.7% of classic IOPD patients and 48.7% of LOPD patients. LOPD patients were more likely to have noninvasive ventilatory support (41.3%) as compared to patients with classic IOPD. In 2014, 82.4% of patients were reported to have ever been on enzyme replacement therapy (ERT) with alglucosidase alfa, 15.8% were never on ERT, and ERT status was unknown in 1.5% of patients. The overall mean age at first infusion of ERT was 7.3 months and 39.5 years in patients with classic IOPD and LOPD, respectively. The Pompe Registry has been critical in identifying new signs and symptoms not traditionally associated with Pompe disease, including arterial aneurysms, lingual weakness, oropharyngeal dysphagia, ptosis, and scoliosis. Conclusions: With the largest enrollment of Pompe patients worldwide, the Pompe Registry continues to be a valuable resource for improving the understanding of Pompe disease in the medical community. The Registry contributes substantially to the clinical understanding of this rare disease and will continue to play a crucial role in collecting, understanding, and disseminating clinical information about Pompe disease both globally and regionally.
Keisuke Kitakazea, Chikako Tasakia, Yasumichi Mizutania, Eiji Sugiyamab, Mariko Ikuoa, Mako Kamiyac, Mitsutoshi Setoub, Yasuteru Uranoc, Kohji Itoha, aThe University of Tokushima, Tokushima, Japan, bHamamatsu University School of Medicine, Hamamatsu, Japan, cThe University of Tokyo, Tokyo, Japan
OF
Priya Kishnania, Yin-Hsiu Chienb, Juan Llerenac, Judy Kempfd, Zsuzsanna Devecserid, Ans van der Ploege, aDuke University Medical Center, Durham, NC, USA, bNational Taiwan University Hospital, Taipei, Taiwan, cFernandes Figueira Institute — FIOCRUZ, Rio de Janeiro, Brazil, d Genzyme, Cambridge, MA, USA, eErasmus MC University Medical Center, Rotterdam, The Netherlands
135 Development of protease-resistant modified human β-hexosaminidase B and evaluation of intracerebroventricular replacement effects on GM2 gangliosidosis model mice
RO
134 The Pompe Registry: 10 years of data
S65
doi:10.1016/j.ymgme.2014.12.137
doi:10.1016/j.ymgme.2014.12.138
136 Molecular basis of sialidosis and its treatment Nilima Kolli, Scott C. Garman, University of Massachusetts Amherst, Amherst, MA, USA
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
Francyne Kubaskia,b, Tsutomu Shimadab, Shunji Tomatsub, Eriko Yasudab, Robert W. Masonb, William G. Mackenzieb, Yuniko Shibatac, Roberto Giuglianid, Seiji Yamaguchie, Yasuyuki Suzukif, Kenji Oriif, Tadao Oriif, aUniversity of Delaware, Newark, DE, USA, bNemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, cCentral Research Lab., R&D Div., Seikagaku Co. Tokyo, Tokyo, Japan, dMedical Genetics Service — Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil, eShimane University, Izumo, Japan, fGifu University, Gifu, Japan Chondroitin 6-sulfate (C6S), a glycosaminoglycan (GAG), is distributed mainly in the growth plates, aorta, and cornea; however, the physiological function of C6S is not fully understood. One of the limitations is that no rapid, accurate quantitative method to measure C6S has been established. Mucopolysaccharidosis IVA and VII (MPS IVA and VII) are caused by the deficiency of N-acetylgalactosamine6-sulfate sulfatase and β-d-glucuronidase, respectively, resulting in accumulation of C6S and other GAG(s). While levels of keratan sulfate (KS), heparan sulfate and dermatan sulfate in samples from MPS patients are well described, this is the first report of quantitative analysis of C6S levels in samples from MPS IVA and VII patients. We developed a method to digest polymeric C6S and measure resultant disaccharides using liquid chromatography tandem mass spectrometry (LC/MS/MS). C6S levels were measured in blood from control subjects and patients with MPS IVA and VII aged from 0 to 58 years of age. We also assayed KS levels in the same samples for comparison with C6S. Levels of C6S in blood decreased with age and were significantly elevated in patients with MPS IVA and VII, compared with age-matched controls. Levels of KS in patients with MPS IVA were also higher than those in age-matched controls, although differences were less pronounced than with C6S. Combining KS and C6S data, discriminated patients with MPS IVA from age-matched control subjects better than either C6S or KS levels alone. This first report showing that blood levels of C6S are quantitatively evaluated in patients with MPS IVA and VII indicates that C6S could be a useful biomarker for these metabolic disorders.
DP
doi:10.1016/j.ymgme.2014.12.139
137 Analysis of C6S/C4S ratio in Morquio syndrome type A patients
138 Chondroitin 6-sulfate as a novel biomarker for mucopolysaccharidosis IVA and VII
RO
Sialidosis is a lysosomal disease caused by mutations in the lysosomal neuraminidase (NEU1). NEU1 is an exosialidase that cleaves terminal sialic acids from glycoproteins and glycolipids in the lysosome and on the cell surface, highlighting its importance in vital cellular functions such as catabolism and signal transduction pathways. Deficiency of NEU1 activity in patients with mutations in Protective Protein/Cathepsin A (PPCA, galactosialidosis) suggests its dependence on PPCA for normal function. In the current study we show that PPCA directly interacts with NEU1 and, contrary to previous reports, we demonstrate that this interaction is not required for the catalytic activity of NEU1, instead it facilitates stabilization of NEU1. We report for the first time the kinetic parameters of the neuraminidase activity of NEU1, which exhibits a kcat of 2.14 min− 1 and kcat/KM of 1.73 mM− 1 min− 1. Using inhibitors to stabilize NEU1, we further present a proof-of-concept study for pharmacological chaperone therapy for the treatment of sialidosis. Thus, we demonstrate that instability of NEU1 outside the lysosomal complex forms the molecular basis of sialidosis, and propose development of therapeutics focused on stabilization of NEU1.
OF
S66
TE
Francyne Kubaskia,b, Tsutomu Shimadab, Robert W. Masonb, Sherlly L. Xieb, Ana C. Brusius-Facchinc, Fernanda Benderc, Aline N. Bochernitsanc, Sandra Leistner_Segalc, Roberto Giuglianic, Shunji Tomatsub, aUniversity of Delaware, Newark, DE, USA, bNemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, cMedical Genetics Service — Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil
UN
CO
RR
EC
Morquio syndrome type A (MPS IVA) is an autosomal recessive disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) which leads to accumulation of keratan sulfate (KS) and chondroitin sulfate (CS). C6S and C4S are known for their properties in cartilage and C6S/C4S ratio might reflect cartilage metabolism. The aim of this study was to evaluate C6S/C4S ratio in 25 Morquio syndrome type A patients. We standardize a protocol for C4S assay by liquid chromatography–tandem mass spectrometry (LC–MSMS) using Chondroitinase ABC to digest C6S and Chondroitinase ACII for C4S in 6460 Triple Quad mass spectrometer (Agilent Technologies). C4S and C6S levels were measured in the blood of 25 patients and 25 aged matched controls from 3.6 to 26 years of age (average age 12.5). We observed higher levels of C4S in younger patients. The controls presented lower rates compared to patients, however the difference was not statistically significant at the 5% level (Wilcoxon signed-rank test). Among the patients five had high rates compared to age matched controls (2.20, 4.26, 4.69, 7.78, 8.76 ng/ml patient's rates × control's rates: 0.33, 0.36, 0.02, 0.29, 0.01 ng/ml). It is interesting to mention that between these patients the genotype analysis (PCR followed by sequencing) identified mutations associated with severe phenotypes (p.R386C/ p.R386C, p.G116S/?, p.S341R/R386C, p.L307P/S341R, p.G116S/ G116S) which might be associated with the higher rates compared to those of controls. Further analysis with a higher number of patients should be performed to elucidate if C6S/C4S ratio is a good biomarker for Morquio syndrome type A patients.
doi:10.1016/j.ymgme.2014.12.140
doi:10.1016/j.ymgme.2014.12.141
139 Di-sulfated keratan sulfate as a novel biomarker for mucopolysaccharidosis IVA Francyne Kubaskia,b, Tsutomu Shimadab, Shunji Tomatsub, Robert W. Masonb, Eriko Yasudab, William G. Mackenzieb, Jobayer Hossainb, Yuniko Shibatac, Adriana M. Montañod, Roberto Giuglianie, Seiji Yamaguchif, Yasuyuki Suzukig, Kenji E. Oriig, Toshiyuki Fukaog, Tadao Oriig, aUniversity of Delaware, Newark, DE, USA, bNemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, cCentral Research Lab., R&D Div., Seikagaku Co. Tokyo, Tokyo, Japan, dSaint Louis University, Newark, MO, USA, eMedical Genetics Service — Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil, fShimane University, Izumo, Japan, gGifu University, Gifu, Japan Keratan sulfate (KS) is known as a storage material in mucopolysaccharidosis IV (MPS IV). However, no detailed analysis has been reported on subclasses of KS: mono-sulfated KS and di-sulfated KS. We established a novel method for mono- and di-sulfated KS using liquid chromatography tandem mass spectrometry and measured both KS levels in various specimens. Di-sulfated KS was dominant in shark cartilage and rat serum, while mono-sulfated KS
GM2 gangliosidoses, including Tay–Sachs disease (TSD) and Sandhoff disease (SD), are neurodegenerative lysosomal diseases (LSD) associated with excessive accumulation of GM2 ganglioside (GM2) in the brain and neurological symptoms. TSD and SD are the lysosomal β-hexosaminidase A (HexA) deficiencies caused by gene mutations in HEXA and HEXB loci, which encode the α- and β-subunit of HexA (αβ heterodimer), respectively. Hence there are no effective treatments for GM2 gangliosidoses, although several therapeutic approaches have been developed including gene therapy, enzyme replacement therapy (ERT), substrate reduction therapy, etc. Previously we constructed a human modified HEXB cDNA encoding altered β-subunit containing partial amino acid sequence of the α-subunit, and succeeded in the production of the modified HexB (modB) by CHO cell line with the GM2-degrading activity in the brain by intracerebroventricular (icv) administration to SD model mice. In this study, we also designed mod2B containing additional α-sequences to resist protease degradation. The recombinant modB and mod2B carrying N-glycans with terminal mannose 6-phosphate (M6P) residues (M6P-modB and -mod2B) were purified with three-step column chromatography, including an affinity chromatography with Phos-Tag resin, which is usually used to capture phosphorylated proteins. Icv injection of M6P-modB caused uptake by parenchymal neural cells, which was processed to the mature form and partially degraded Hex subunits. In contrast, M6P-mod2B was resistant to neural proteases and maintained the mature form and the GM2-degrading activity. These results suggest that α-sequence around GSEP loop should regulate the resistance to neural proteases. Next, we visualized the delivery of M6P-modB and -mod2B to lysosomes in the target cells derived from TSD and SD model through binding with cell surface cation-independent M6P receptors (CI-M6PR) with novel acidic pHactivatable fluorescent probes, including RhPM as a tag conjugated to modified Hex proteins. We also developed an imaging mass spectrometry (IMS) system to evaluate the distribution and reduction of GM2 and asialo GM2 (GA2) accumulated in the brain sections of SD mice on icv administration. In the brains of untreated SD mice, signals of GM2 (d18:1/18:0) were prominent in the hypothalamus (HTH) and lateral septal nucleus (LSN). GM2 (d18:1/20:0) were observed especially, in the cerebellum (CB). Signals of GA2 (d18:1/18:0) and GA2 (d18:1/20:0) were observed throughout the brain. On treatment with M6P-mod2B, compared to M6P-modB, signals of GM2 and GA2 were remarkably decreased in the whole area including cerebral cortex (CCX), CB, HTH and LSN. Furthermore, icv administration of M6P-mod2B has remarkable therapeutic effects on SD mice, including improvement of motor dysfunction. These results suggest that novel intracerebroventricular ERT with protease-resistant modified human β-hexosaminidase B (M6Pmod2B) should be applicable for patients with GM2 gangliosidoses.
UN
CO
RR
EC
TE
DP
Background: In 2014, the Pompe Registry is recognizing and celebrating an important milestone: the 10-year anniversary of the Registry. Methods: Started in 2004 and sponsored by Genzyme, a Sanofi company, the Pompe Registry was established as a global, voluntary, observational database to collect and report on the natural history and outcomes of patients with Pompe disease through routine clinical practice, regardless of patient treatment status. Data currently are collected and reported by enrolling clinicians worldwide in four geographic regions: Europe, North America, Latin America, and the Asia Pacific region. Results: As of May 2014, 1292 patients in 34 countries were enrolled in the Registry. The majority (70.1%) was Caucasian, and there was an approximate equal distribution of males and females. More than three-quarters (78.9%) of enrolled patients had Late-onset Pompe disease (LOPD), defined as symptom onset ≤12 months of age without cardiomyopathy or symptom onset N12 months of age; 13.8% of patients had classic Infantile-onset Pompe disease (IOPD), defined as symptom onset ≤12 months with cardiomyopathy. Age at symptom onset was unknown in 7.3% of patients. In classic IOPD, the overall mean age at symptom onset was 2.9 months in 2014. The mean age at diagnosis for these patients was 4.0 months, signifying a diagnostic gap of 1.1 months. In 2014, the overall mean age of symptom onset and diagnosis for LOPD patients was 26.5 years and 33.5 years, respectively. Blood-based enzyme assays that measure acid alpha-glucosidase (GAA) activity (including dried blood spots [DBS], leukocytes, and lymphocytes), and DNA analysis were the most frequently used methods of diagnosis for patients in the Pompe Registry. Among classic IOPD patients, hypotonia was the most frequently reported symptom overall, affecting 94.7% of patients. Ambulation was impaired in 78.4% of LOPD patients in the Registry. Two-thirds (67.6%) of LOPD patients reported to have lost ambulatory capabilities, and approximately half reported using ambulation devices. Overall, 47.1% of all Registry patients had reported receiving respiratory support of some kind: 43.7% of classic IOPD patients and 48.7% of LOPD patients. LOPD patients were more likely to have noninvasive ventilatory support (41.3%) as compared to patients with classic IOPD. In 2014, 82.4% of patients were reported to have ever been on enzyme replacement therapy (ERT) with alglucosidase alfa, 15.8% were never on ERT, and ERT status was unknown in 1.5% of patients. The overall mean age at first infusion of ERT was 7.3 months and 39.5 years in patients with classic IOPD and LOPD, respectively. The Pompe Registry has been critical in identifying new signs and symptoms not traditionally associated with Pompe disease, including arterial aneurysms, lingual weakness, oropharyngeal dysphagia, ptosis, and scoliosis. Conclusions: With the largest enrollment of Pompe patients worldwide, the Pompe Registry continues to be a valuable resource for improving the understanding of Pompe disease in the medical community. The Registry contributes substantially to the clinical understanding of this rare disease and will continue to play a crucial role in collecting, understanding, and disseminating clinical information about Pompe disease both globally and regionally.
Keisuke Kitakazea, Chikako Tasakia, Yasumichi Mizutania, Eiji Sugiyamab, Mariko Ikuoa, Mako Kamiyac, Mitsutoshi Setoub, Yasuteru Uranoc, Kohji Itoha, aThe University of Tokushima, Tokushima, Japan, bHamamatsu University School of Medicine, Hamamatsu, Japan, cThe University of Tokyo, Tokyo, Japan
OF
Priya Kishnania, Yin-Hsiu Chienb, Juan Llerenac, Judy Kempfd, Zsuzsanna Devecserid, Ans van der Ploege, aDuke University Medical Center, Durham, NC, USA, bNational Taiwan University Hospital, Taipei, Taiwan, cFernandes Figueira Institute — FIOCRUZ, Rio de Janeiro, Brazil, d Genzyme, Cambridge, MA, USA, eErasmus MC University Medical Center, Rotterdam, The Netherlands
135 Development of protease-resistant modified human β-hexosaminidase B and evaluation of intracerebroventricular replacement effects on GM2 gangliosidosis model mice
RO
134 The Pompe Registry: 10 years of data
S65
doi:10.1016/j.ymgme.2014.12.137
doi:10.1016/j.ymgme.2014.12.138
136 Molecular basis of sialidosis and its treatment Nilima Kolli, Scott C. Garman, University of Massachusetts Amherst, Amherst, MA, USA
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
Francyne Kubaskia,b, Tsutomu Shimadab, Shunji Tomatsub, Eriko Yasudab, Robert W. Masonb, William G. Mackenzieb, Yuniko Shibatac, Roberto Giuglianid, Seiji Yamaguchie, Yasuyuki Suzukif, Kenji Oriif, Tadao Oriif, aUniversity of Delaware, Newark, DE, USA, bNemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, cCentral Research Lab., R&D Div., Seikagaku Co. Tokyo, Tokyo, Japan, dMedical Genetics Service — Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil, eShimane University, Izumo, Japan, fGifu University, Gifu, Japan Chondroitin 6-sulfate (C6S), a glycosaminoglycan (GAG), is distributed mainly in the growth plates, aorta, and cornea; however, the physiological function of C6S is not fully understood. One of the limitations is that no rapid, accurate quantitative method to measure C6S has been established. Mucopolysaccharidosis IVA and VII (MPS IVA and VII) are caused by the deficiency of N-acetylgalactosamine6-sulfate sulfatase and β-d-glucuronidase, respectively, resulting in accumulation of C6S and other GAG(s). While levels of keratan sulfate (KS), heparan sulfate and dermatan sulfate in samples from MPS patients are well described, this is the first report of quantitative analysis of C6S levels in samples from MPS IVA and VII patients. We developed a method to digest polymeric C6S and measure resultant disaccharides using liquid chromatography tandem mass spectrometry (LC/MS/MS). C6S levels were measured in blood from control subjects and patients with MPS IVA and VII aged from 0 to 58 years of age. We also assayed KS levels in the same samples for comparison with C6S. Levels of C6S in blood decreased with age and were significantly elevated in patients with MPS IVA and VII, compared with age-matched controls. Levels of KS in patients with MPS IVA were also higher than those in age-matched controls, although differences were less pronounced than with C6S. Combining KS and C6S data, discriminated patients with MPS IVA from age-matched control subjects better than either C6S or KS levels alone. This first report showing that blood levels of C6S are quantitatively evaluated in patients with MPS IVA and VII indicates that C6S could be a useful biomarker for these metabolic disorders.
DP
doi:10.1016/j.ymgme.2014.12.139
137 Analysis of C6S/C4S ratio in Morquio syndrome type A patients
138 Chondroitin 6-sulfate as a novel biomarker for mucopolysaccharidosis IVA and VII
RO
Sialidosis is a lysosomal disease caused by mutations in the lysosomal neuraminidase (NEU1). NEU1 is an exosialidase that cleaves terminal sialic acids from glycoproteins and glycolipids in the lysosome and on the cell surface, highlighting its importance in vital cellular functions such as catabolism and signal transduction pathways. Deficiency of NEU1 activity in patients with mutations in Protective Protein/Cathepsin A (PPCA, galactosialidosis) suggests its dependence on PPCA for normal function. In the current study we show that PPCA directly interacts with NEU1 and, contrary to previous reports, we demonstrate that this interaction is not required for the catalytic activity of NEU1, instead it facilitates stabilization of NEU1. We report for the first time the kinetic parameters of the neuraminidase activity of NEU1, which exhibits a kcat of 2.14 min− 1 and kcat/KM of 1.73 mM− 1 min− 1. Using inhibitors to stabilize NEU1, we further present a proof-of-concept study for pharmacological chaperone therapy for the treatment of sialidosis. Thus, we demonstrate that instability of NEU1 outside the lysosomal complex forms the molecular basis of sialidosis, and propose development of therapeutics focused on stabilization of NEU1.
OF
S66
TE
Francyne Kubaskia,b, Tsutomu Shimadab, Robert W. Masonb, Sherlly L. Xieb, Ana C. Brusius-Facchinc, Fernanda Benderc, Aline N. Bochernitsanc, Sandra Leistner_Segalc, Roberto Giuglianic, Shunji Tomatsub, aUniversity of Delaware, Newark, DE, USA, bNemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, cMedical Genetics Service — Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil
UN
CO
RR
EC
Morquio syndrome type A (MPS IVA) is an autosomal recessive disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) which leads to accumulation of keratan sulfate (KS) and chondroitin sulfate (CS). C6S and C4S are known for their properties in cartilage and C6S/C4S ratio might reflect cartilage metabolism. The aim of this study was to evaluate C6S/C4S ratio in 25 Morquio syndrome type A patients. We standardize a protocol for C4S assay by liquid chromatography–tandem mass spectrometry (LC–MSMS) using Chondroitinase ABC to digest C6S and Chondroitinase ACII for C4S in 6460 Triple Quad mass spectrometer (Agilent Technologies). C4S and C6S levels were measured in the blood of 25 patients and 25 aged matched controls from 3.6 to 26 years of age (average age 12.5). We observed higher levels of C4S in younger patients. The controls presented lower rates compared to patients, however the difference was not statistically significant at the 5% level (Wilcoxon signed-rank test). Among the patients five had high rates compared to age matched controls (2.20, 4.26, 4.69, 7.78, 8.76 ng/ml patient's rates × control's rates: 0.33, 0.36, 0.02, 0.29, 0.01 ng/ml). It is interesting to mention that between these patients the genotype analysis (PCR followed by sequencing) identified mutations associated with severe phenotypes (p.R386C/ p.R386C, p.G116S/?, p.S341R/R386C, p.L307P/S341R, p.G116S/ G116S) which might be associated with the higher rates compared to those of controls. Further analysis with a higher number of patients should be performed to elucidate if C6S/C4S ratio is a good biomarker for Morquio syndrome type A patients.
doi:10.1016/j.ymgme.2014.12.140
doi:10.1016/j.ymgme.2014.12.141
139 Di-sulfated keratan sulfate as a novel biomarker for mucopolysaccharidosis IVA Francyne Kubaskia,b, Tsutomu Shimadab, Shunji Tomatsub, Robert W. Masonb, Eriko Yasudab, William G. Mackenzieb, Jobayer Hossainb, Yuniko Shibatac, Adriana M. Montañod, Roberto Giuglianie, Seiji Yamaguchif, Yasuyuki Suzukig, Kenji E. Oriig, Toshiyuki Fukaog, Tadao Oriig, aUniversity of Delaware, Newark, DE, USA, bNemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, cCentral Research Lab., R&D Div., Seikagaku Co. Tokyo, Tokyo, Japan, dSaint Louis University, Newark, MO, USA, eMedical Genetics Service — Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil, fShimane University, Izumo, Japan, gGifu University, Gifu, Japan Keratan sulfate (KS) is known as a storage material in mucopolysaccharidosis IV (MPS IV). However, no detailed analysis has been reported on subclasses of KS: mono-sulfated KS and di-sulfated KS. We established a novel method for mono- and di-sulfated KS using liquid chromatography tandem mass spectrometry and measured both KS levels in various specimens. Di-sulfated KS was dominant in shark cartilage and rat serum, while mono-sulfated KS