- Email: [email protected]
Thromboembolic events in Fabry disease and the impact of factor V Leiden
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
(3) already known mutations of Morquio syndrome type A disease. We evaluated predictions of the immunogenicity, post-translational modifications, physico-chemical properties, and 3D structural evaluation of molecular docking. In silico analyses were performed in the new sequences harboring various combinations of substitutions on various immunodominant peptide regions within the whole GALNS protein. In vitro experiments were conducted on HEK 293 cells to determine enzyme activity of the mutated GALNS proteins. 324 mutated sequences were created and narrowed to 7 sequences after analyzing (1) the immunogenicity predictions, (2) the predictions of phosphorylation sites, N-glycosylation sites, and physico-chemical properties, and (3) three-dimensional visualization of molecular docking. Sequences were selected if the altered sequence showed zero or insignificant changes when compared to the original GALNS sequence. The distances between the active site residues and the corresponding ligand were measured in angstroms. Mutated sequences were chosen if their measured distances within the active site were very close to or even smaller than the measured distance in the original GALNS sequence, indicating a greater likelihood to bind. The cDNA sequence was used for the determination of restriction sites and mutagenesis of the 7 selected sequences. Three out of seven mutated GALNS sequences had 80% or more enzyme activity compared to the wild type GALNS protein in vitro. In conclusion, a less immunogenic GALNS would improve the efficacy of ERT, remove the two to three year period in which the patient develops tolerance to the protein, and ultimately eliminate the need for immunosuppressive protocols of ERT.
DP
RO
OF
2014 the S4RD team was featured in a national newspaper and this year the Royal Society of Medicine will host the 3rd symposium. There will be speakers covering Tay Sachs and Pompe disease. S4RD hopes to encourage all UK Medical Schools to adopt this model, to help raise awareness of rare diseases in future generations of clinicians. Furthermore timely diagnosis and treatment of rare diseases is a global issue but with online resources and social media it would be possible for international collaboration between medical students. Educating future doctors on this topic is essential for improving the challenges faced by rare patients and their families, and help the 1 in 17 achieve fast diagnosis and full medical support.
S71
doi:10.1016/j.ymgme.2014.12.152
TE
150 Thromboembolic events in Fabry disease and the impact of factor V Leiden
RR
doi:10.1016/j.ymgme.2014.12.151
EC
Malte Lendersa, Nesrin Karabulb, Thomas Duninga, Boris Schmitza, Michael Schelleckesa, Rolf Mestersa, Hans-Werner Hensea, Michael Beckb, Stefan-Martin Branda, Eva Branda, aUniverity Hopital Muenster, Muenster, Germany, bUniversity of Mainz, Mainz, Germany
CO
149 Development of a less immunogenic protein for enzyme replacement therapy of Morquio syndrome type A disease Alexandria A. Lee, Angela C. Sosa, Adriana M. Montaño, Saint Louis University, St Louis, MO, USA
UN
Immune response against proteins used for enzyme replacement therapy (ERT) has been characterized as one of the main limitations in the treatment effectiveness. Preclinical trials of enzyme replacement therapy (ERT) in mucopolysaccharidosis IVA (Morquio syndrome type A disease, MPS IVA) have shown that ERT reduces accumulation of glycosaminoglycans, but is accompanied by a large immune response that significantly decreases the efficacy of this treatment. The aim of this study is to bioengineer a human GALNS protein with reduced immunogenicity without affecting the biological activity for effective ERT of MPS IVA. The original GALNS amino acid and cDNA sequences were compared to the corresponding altered sequences in a variety of in silico programs. Upon creating different combinations of amino acid substitutions, considerations included (1) polarity, (2) amino acids with unique structures, and
Although several reports suggest an increased thromboembolic event rate, especially with respect to strokes and transient ischemic attacks (TIA) at early age in patients with Fabry disease (FD), the risk for Fabry patients to suffer from these events, the clinical relevance of additional risk factors including the concurrence of Factor V Leiden (FVL) and the benefit of enzyme replacement therapy (ERT) regarding these events remains unclear. We evaluated 304 consecutively recruited Fabry patients for their life-time occurrence of thromboembolic events such as stroke, TIA, deep vein thrombosis and pulmonary embolism. The thromboembolic risk was determined in Fabry patients with concurrent FVL, and the impact of ERT was assessed. The 304 Fabry patients had a median age of 41 years and 53 (17.4%) had suffered at least one thromboembolic event during their lifetime. Among 226 Fabry patients screened for FVL, 16 gene carriers were identified (7.1%). The occurrence of thromboembolic events in Fabry patients with concurrent FVL was significantly increased compared to those without FVL (HR = 5.45, 95% CI [2.29–12.99]; p b 0.001). Fabry patients under ERT had a significantly decreased risk for thromboembolic events compared to those without ERT (HR = 0.362, 95% CI [0.132–0.992]; p = 0.0422). This observational study confirms that Fabry patients suffer from a high risk of clinically relevant thromboembolic events, which could be aggravated by a concurrence of FVL. ERT might be of benefit in preventing vascular events in patients with Fabry disease. The latter observation needs confirmation, however, by randomized and controlled clinical trials.
S72
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
de-identified. A study number will link identifiers to the deidentified data. This key will be held at the coordinating center, with access only by the coordinating center's PI and a data manager. Our natural history study will fill a gap in the understanding of Sanfilippo syndrome (MPS III), as prior studies focused on the more common MPS IIIA and MPS IIIB. We will describe our experiences developing the protocol in detail along with inclusion and exclusion criteria. This should be useful information for others wishing to develop their own NHS in future. We should begin to enroll patients by December 2014 or early 2015. doi:10.1016/j.ymgme.2014.12.154
OF
152 Two masters of lysosomal and autophagosomal biogenesis, TFEB and TFE3, and their potential therapeutic value in Pompe disease doi:10.1016/j.ymgme.2014.12.153
151 Experiences from setting up a 5 year longitudinal, prospective, natural history study of patients with Sanfilippo syndrome types C or D (MPS IIIC or MPS IIID)
Pompe disease is a lysosomal glycogen storage disorder (glycogenosis type II) caused by a deficiency of acid α-glucosidase (GAA). In this severe myopathy, lysosomal glycogen accumulation leads to defective autophagic flux, a process of autophagosome formation, fusion with the lysosome, and degradation of autophagic substrates inside the lysosome. Inhibition of the autophagic flux in Pompe skeletal muscle results in autophagic buildup that disturbs sarcomere structure and negatively affects vesicular trafficking and the delivery of the replacement enzyme (recombinant human GAA) to the lysosomes. Our recent studies have focused on the development of new approaches designed to re-establish proper autophagic flux in affected muscles. Overexpression of transcription factor EB (TFEB; a master regulator of lysosomal and autophagosomal biogenesis) in myotubes and myofibers triggered exocytosis of enlarged-glycogen filled lysosomes, increased fusion of lysosomes and autophagosomes, and alleviated excessive accumulation of autophagic vacuoles in both Pompe cellular and mouse models. These data established TFEB as a viable therapeutic target in Pompe disease. More recently we have shown that a closely related but distinct transcription factor E3 (TFE3) is a more attractive target since it is abundant in skeletal muscle, whereas TFEB is not. We have used ChIPseq to identify the genome-wide distribution of TFE3 sites in muscle cells; the inspection of mapped peak sets established a remarkable similarity to known TFEB binding locations. Like TFEB, overexpression of TFE3 in the diseased muscle cells induced lysosomal exocytosis, dramatically reduced the lysosomal size and cleared the cells of excess glycogen. As with TFEB, TFE3 activity is regulated by phosphorylation; inhibition of phosphorylation activates TFE3 and promotes its translocation to the nucleus where it stimulates the expression of multiple target genes. Therefore, pharmacological inhibition of TFE3 phosphorylation would promote cellular clearance in Pompe disease as well as in other lysosomal disorders.
DP
Paul A. Levya, Jill Woodb, aEinstein/Montefiore, Bronx, NY, USA, bJonah's Just Begun, Brooklyn, NY, USA
RO
Lishu Li, Jeong-A Lim, Nina Raben, NIAMS, National Institutes of Health, Bethesda, MD, USA
UN
CO
RR
EC
TE
The overall aim of this project is to expand our knowledge of the clinical features of MPS IIIC and MPS IIID disease by carrying out a prospective natural history study (NHS) for a period of 5 years. With the help of Jonah's Just Begun (JJB) a Sanfilippo parent advocacy and research group, 15 patients with MPS IIIC and 2 patients with MPS IIID have been identified. JJB is a sponsor of the study and they wish for the data collected to be freely available to other researchers. During the course of the study, we will determine the genotype and the residual enzyme activity, as well as do periodic measurements by neurocognitive testing, measure glycosaminoglycans in urine, do observations of clinical status, and use brain imaging to document the course of the disease. Our hypothesis is that disease progression will be manifested by neurocognitive decline, and will correlate with genotype and/or residual enzyme activity, as well as with an increase in glycosaminoglycan levels in urine. Aim 1: Document the clinical course of the disease. A. We will use standard neuropsychological testing to document the cognitive status of patients and correlate this with their disease status. Cognitive regression over time will be documented by following longitudinal changes of the neuropsychological testing. B. We will use MRI to observe changes over time of brain structure, myelination, ventricular size and cortical atrophy as have been reported in previous studies. We will also use brain volumetrics and DTI to obtain measurements of these changes over time in patients with Sanfilippo IIIC and IIID. Aim 2: Evaluate biomarkers as correlates to disease status A. We will measure residual enzyme activity (in blood leukocytes or fibroblasts) and attempt to correlate the findings with genotype and disease progression with time. B. We will measure urinary glycosaminoglycans and attempt to correlate these changes with disease progression7. Aim 3: Compare our data with other studies of Sanfilippo patients to help better delineate the phenotype and differences between types IIIC and IIID with IIIA and IIIB. As part of the Lysosomal Disease Network — a Rare Diseases Clinical Center Research Network (LDN RDCRN), data from the natural history study will be entered into a secure database at the Data Management Coordinating Center (DMCC). The patient data are intended to be used as a resource for other researchers. Data that are entered will be
doi:10.1016/j.ymgme.2014.12.155
153 Pathogenetic studies of 13 novel missense mutations in α-galactosidase A from the newborn screening in Taiwan HsuanChieh Liaoa, Yann-Jang Chenb, Ting-Rong Hsua, Chuan-Chi Chiangc, Dau-Ming Niua, aInstitute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, bInstitute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, cChinese Foundation of Health, Taipei, Taiwan
(3) already known mutations of Morquio syndrome type A disease. We evaluated predictions of the immunogenicity, post-translational modifications, physico-chemical properties, and 3D structural evaluation of molecular docking. In silico analyses were performed in the new sequences harboring various combinations of substitutions on various immunodominant peptide regions within the whole GALNS protein. In vitro experiments were conducted on HEK 293 cells to determine enzyme activity of the mutated GALNS proteins. 324 mutated sequences were created and narrowed to 7 sequences after analyzing (1) the immunogenicity predictions, (2) the predictions of phosphorylation sites, N-glycosylation sites, and physico-chemical properties, and (3) three-dimensional visualization of molecular docking. Sequences were selected if the altered sequence showed zero or insignificant changes when compared to the original GALNS sequence. The distances between the active site residues and the corresponding ligand were measured in angstroms. Mutated sequences were chosen if their measured distances within the active site were very close to or even smaller than the measured distance in the original GALNS sequence, indicating a greater likelihood to bind. The cDNA sequence was used for the determination of restriction sites and mutagenesis of the 7 selected sequences. Three out of seven mutated GALNS sequences had 80% or more enzyme activity compared to the wild type GALNS protein in vitro. In conclusion, a less immunogenic GALNS would improve the efficacy of ERT, remove the two to three year period in which the patient develops tolerance to the protein, and ultimately eliminate the need for immunosuppressive protocols of ERT.
DP
RO
OF
2014 the S4RD team was featured in a national newspaper and this year the Royal Society of Medicine will host the 3rd symposium. There will be speakers covering Tay Sachs and Pompe disease. S4RD hopes to encourage all UK Medical Schools to adopt this model, to help raise awareness of rare diseases in future generations of clinicians. Furthermore timely diagnosis and treatment of rare diseases is a global issue but with online resources and social media it would be possible for international collaboration between medical students. Educating future doctors on this topic is essential for improving the challenges faced by rare patients and their families, and help the 1 in 17 achieve fast diagnosis and full medical support.
S71
doi:10.1016/j.ymgme.2014.12.152
TE
150 Thromboembolic events in Fabry disease and the impact of factor V Leiden
RR
doi:10.1016/j.ymgme.2014.12.151
EC
Malte Lendersa, Nesrin Karabulb, Thomas Duninga, Boris Schmitza, Michael Schelleckesa, Rolf Mestersa, Hans-Werner Hensea, Michael Beckb, Stefan-Martin Branda, Eva Branda, aUniverity Hopital Muenster, Muenster, Germany, bUniversity of Mainz, Mainz, Germany
CO
149 Development of a less immunogenic protein for enzyme replacement therapy of Morquio syndrome type A disease Alexandria A. Lee, Angela C. Sosa, Adriana M. Montaño, Saint Louis University, St Louis, MO, USA
UN
Immune response against proteins used for enzyme replacement therapy (ERT) has been characterized as one of the main limitations in the treatment effectiveness. Preclinical trials of enzyme replacement therapy (ERT) in mucopolysaccharidosis IVA (Morquio syndrome type A disease, MPS IVA) have shown that ERT reduces accumulation of glycosaminoglycans, but is accompanied by a large immune response that significantly decreases the efficacy of this treatment. The aim of this study is to bioengineer a human GALNS protein with reduced immunogenicity without affecting the biological activity for effective ERT of MPS IVA. The original GALNS amino acid and cDNA sequences were compared to the corresponding altered sequences in a variety of in silico programs. Upon creating different combinations of amino acid substitutions, considerations included (1) polarity, (2) amino acids with unique structures, and
Although several reports suggest an increased thromboembolic event rate, especially with respect to strokes and transient ischemic attacks (TIA) at early age in patients with Fabry disease (FD), the risk for Fabry patients to suffer from these events, the clinical relevance of additional risk factors including the concurrence of Factor V Leiden (FVL) and the benefit of enzyme replacement therapy (ERT) regarding these events remains unclear. We evaluated 304 consecutively recruited Fabry patients for their life-time occurrence of thromboembolic events such as stroke, TIA, deep vein thrombosis and pulmonary embolism. The thromboembolic risk was determined in Fabry patients with concurrent FVL, and the impact of ERT was assessed. The 304 Fabry patients had a median age of 41 years and 53 (17.4%) had suffered at least one thromboembolic event during their lifetime. Among 226 Fabry patients screened for FVL, 16 gene carriers were identified (7.1%). The occurrence of thromboembolic events in Fabry patients with concurrent FVL was significantly increased compared to those without FVL (HR = 5.45, 95% CI [2.29–12.99]; p b 0.001). Fabry patients under ERT had a significantly decreased risk for thromboembolic events compared to those without ERT (HR = 0.362, 95% CI [0.132–0.992]; p = 0.0422). This observational study confirms that Fabry patients suffer from a high risk of clinically relevant thromboembolic events, which could be aggravated by a concurrence of FVL. ERT might be of benefit in preventing vascular events in patients with Fabry disease. The latter observation needs confirmation, however, by randomized and controlled clinical trials.
S72
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
de-identified. A study number will link identifiers to the deidentified data. This key will be held at the coordinating center, with access only by the coordinating center's PI and a data manager. Our natural history study will fill a gap in the understanding of Sanfilippo syndrome (MPS III), as prior studies focused on the more common MPS IIIA and MPS IIIB. We will describe our experiences developing the protocol in detail along with inclusion and exclusion criteria. This should be useful information for others wishing to develop their own NHS in future. We should begin to enroll patients by December 2014 or early 2015. doi:10.1016/j.ymgme.2014.12.154
OF
152 Two masters of lysosomal and autophagosomal biogenesis, TFEB and TFE3, and their potential therapeutic value in Pompe disease doi:10.1016/j.ymgme.2014.12.153
151 Experiences from setting up a 5 year longitudinal, prospective, natural history study of patients with Sanfilippo syndrome types C or D (MPS IIIC or MPS IIID)
Pompe disease is a lysosomal glycogen storage disorder (glycogenosis type II) caused by a deficiency of acid α-glucosidase (GAA). In this severe myopathy, lysosomal glycogen accumulation leads to defective autophagic flux, a process of autophagosome formation, fusion with the lysosome, and degradation of autophagic substrates inside the lysosome. Inhibition of the autophagic flux in Pompe skeletal muscle results in autophagic buildup that disturbs sarcomere structure and negatively affects vesicular trafficking and the delivery of the replacement enzyme (recombinant human GAA) to the lysosomes. Our recent studies have focused on the development of new approaches designed to re-establish proper autophagic flux in affected muscles. Overexpression of transcription factor EB (TFEB; a master regulator of lysosomal and autophagosomal biogenesis) in myotubes and myofibers triggered exocytosis of enlarged-glycogen filled lysosomes, increased fusion of lysosomes and autophagosomes, and alleviated excessive accumulation of autophagic vacuoles in both Pompe cellular and mouse models. These data established TFEB as a viable therapeutic target in Pompe disease. More recently we have shown that a closely related but distinct transcription factor E3 (TFE3) is a more attractive target since it is abundant in skeletal muscle, whereas TFEB is not. We have used ChIPseq to identify the genome-wide distribution of TFE3 sites in muscle cells; the inspection of mapped peak sets established a remarkable similarity to known TFEB binding locations. Like TFEB, overexpression of TFE3 in the diseased muscle cells induced lysosomal exocytosis, dramatically reduced the lysosomal size and cleared the cells of excess glycogen. As with TFEB, TFE3 activity is regulated by phosphorylation; inhibition of phosphorylation activates TFE3 and promotes its translocation to the nucleus where it stimulates the expression of multiple target genes. Therefore, pharmacological inhibition of TFE3 phosphorylation would promote cellular clearance in Pompe disease as well as in other lysosomal disorders.
DP
Paul A. Levya, Jill Woodb, aEinstein/Montefiore, Bronx, NY, USA, bJonah's Just Begun, Brooklyn, NY, USA
RO
Lishu Li, Jeong-A Lim, Nina Raben, NIAMS, National Institutes of Health, Bethesda, MD, USA
UN
CO
RR
EC
TE
The overall aim of this project is to expand our knowledge of the clinical features of MPS IIIC and MPS IIID disease by carrying out a prospective natural history study (NHS) for a period of 5 years. With the help of Jonah's Just Begun (JJB) a Sanfilippo parent advocacy and research group, 15 patients with MPS IIIC and 2 patients with MPS IIID have been identified. JJB is a sponsor of the study and they wish for the data collected to be freely available to other researchers. During the course of the study, we will determine the genotype and the residual enzyme activity, as well as do periodic measurements by neurocognitive testing, measure glycosaminoglycans in urine, do observations of clinical status, and use brain imaging to document the course of the disease. Our hypothesis is that disease progression will be manifested by neurocognitive decline, and will correlate with genotype and/or residual enzyme activity, as well as with an increase in glycosaminoglycan levels in urine. Aim 1: Document the clinical course of the disease. A. We will use standard neuropsychological testing to document the cognitive status of patients and correlate this with their disease status. Cognitive regression over time will be documented by following longitudinal changes of the neuropsychological testing. B. We will use MRI to observe changes over time of brain structure, myelination, ventricular size and cortical atrophy as have been reported in previous studies. We will also use brain volumetrics and DTI to obtain measurements of these changes over time in patients with Sanfilippo IIIC and IIID. Aim 2: Evaluate biomarkers as correlates to disease status A. We will measure residual enzyme activity (in blood leukocytes or fibroblasts) and attempt to correlate the findings with genotype and disease progression with time. B. We will measure urinary glycosaminoglycans and attempt to correlate these changes with disease progression7. Aim 3: Compare our data with other studies of Sanfilippo patients to help better delineate the phenotype and differences between types IIIC and IIID with IIIA and IIIB. As part of the Lysosomal Disease Network — a Rare Diseases Clinical Center Research Network (LDN RDCRN), data from the natural history study will be entered into a secure database at the Data Management Coordinating Center (DMCC). The patient data are intended to be used as a resource for other researchers. Data that are entered will be
doi:10.1016/j.ymgme.2014.12.155
153 Pathogenetic studies of 13 novel missense mutations in α-galactosidase A from the newborn screening in Taiwan HsuanChieh Liaoa, Yann-Jang Chenb, Ting-Rong Hsua, Chuan-Chi Chiangc, Dau-Ming Niua, aInstitute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, bInstitute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, cChinese Foundation of Health, Taipei, Taiwan